Effects of a 12-week high-α-linolenic acid intervention on EPA and DHA concentrations in red blood cells and plasma oxylipin pattern in subjects with a low EPA and DHA status†

The essential omega-3 fatty acid alpha-linolenic acid (ALA, 18:3n3) can be converted into EPA and DHA. The aim of the present study was to determine the effect of a high-ALA diet on EPA and DHA levels in red blood cells (RBCs) and their oxylipins in the plasma of subjects with a low EPA and DHA status. Fatty acid concentrations [μg mL] and relative amounts [% of total fatty acids] in the RBCs of 19 healthy men (mean age 26.4 ± 4.6 years) were analyzed by means of GC-FID. Free plasma oxylipin concentrations were determined by LC-MS based targeted metabolomics. Samples were collected and analyzed at baseline (week 0) and after 1 (week 1), 3 (week 3), 6 (week 6), and 12 (week 12) weeks of high dietary ALA intake (14.0 ± 0.45 g day). ALA concentrations significantly (p < 0.001) increased from 1.44 ± 0.10 (week 0) to 4.65 ± 0.22 (week 1), 5.47 ± 0.23 (week 3), 6.25 ± 0.24 (week 6), and 5.80 ± 0.28 (week 12) μg mL. EPA concentrations increased from 6.13 ± 0.51 (week 0) to 7.33 ± 0.33 (week 1), 8.38 ± 0.42 (p = 0.021, week 3), 10.9 ± 0.67 (p < 0.001, week 6), and 11.0 ± 0.64 (p < 0.001, week 12) μg mL. DHA concentrations unexpectedly decreased from 41.0 ± 1.93 (week 0) to 37.0 ± 1.32 (week 1), 36.1 ± 1.37 (week 3), 35.1 ± 1.06 (p = 0.010, week 6), and 30.4 ± 1.09 (p < 0.001, week 12) μg mL. Relative ΣEPA + DHA amounts were unchanged during the intervention (week 0: 4.63 ± 0.19, week 1: 4.67 ± 0.16, week 3: 4.61 ± 0.13, week 6: 4.73 ± 0.15, week 12: 4.52 ± 0.11). ALAand EPA-derived hydroxyand dihydroxyPUFA increased similarly to their PUFA precursors, although in the case of ALA-derived oxylipins, the concentrations increased less rapidly and to a lesser extent compared to the concentrations of their precursor FA. LA-derived oxylipins remained unchanged and arachidonic acid and DHA oxylipin concentrations were not significantly changed. Our results confirm that the intake of ALA is not a sufficient source for the increase of EPA + DHA in subjects on a Western diet. Specifically, a high-ALA diet results in increased EPA and declined DHA concentrations. However, the changes effectively balance each other out so that ΣEPA + DHA in RBCs – which is an established marker for health protective effects of omega-3-PUFA – remains constant. The PUFA levels in RBCs reflect the concentration and its changes in plasma hydroxyand dihydroxy-PUFA concentrations for ALA and EPA.


Introduction
Health benefits including lower risk of cardiovascular disease and mortality, 1-5 reduction of inflammatory conditions, 2,6 angiogenesis, tumor growth and metastasis 2,7 and a better visual and neurological development 8 are associated with long chain (LC) omega-3 (n3) polyunsaturated fatty acids (PUFAs), namely eicosapentaenoic acid (EPA, C20:5n3) and docosahexaenoic acid (DHA, C22:6n3). The positive health effects are believed to be mediated in part by the oxylipins formed from EPA and DHA in the arachidonic acid (AA, C20:4n6) cascade via different enzymatic pathways: cyclooxygenase (COX) action leads to prostanoid formation, lipoxygenases (LOX) give rise to concentrations in red blood cells (RBCs) has not been studied in a long-term study with healthy omnivores.
The aim of the present study was therefore to determine the short (1 and 3 weeks) and long term (6 and 12 weeks) effect of a high-ALA diet providing a daily ALA dose of 14.0 ± 0.45 g on EPA and DHA concentrations and relative EPA and DHA amounts in RBCs and their oxylipins in the plasma of subjects with a low EPA and DHA status consuming a Western (German) diet.

Materials and methods
This investigator initiated study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects were approved by the ethics committee at the medical chamber of Lower Saxony (Hannover, Germany). Written informed consent was obtained from all subjects. The study is registered in the German clinical trial register (no. DRKS00006765).

Study design
The study was conducted at the Institute of Food Science and Human Nutrition, Leibniz University Hannover, Germany and consisted of a screening phase, a 12-week lasting intervention period and an 8-week lasting follow-up period. During the intervention period, five examinations were carried out, at the beginning (week 0) and after one (week 1), three (week 3), six (week 6) and twelve (week 12) weeks. Two further examinations were carried out in the follow-up period (week 14 and week 20). During the intervention period, the subjects daily ingested 22.3 g of linseed oil (All Organic Trading GmbH, Kempten, Germany) with an ALA content of 58% of total fatty acids (Table S1 †). Hence, the ALA intake from linseed oil was 12.9 g day −1 . The peroxide value of the linseed oil was 1.0 meq kg −1 and the acid value was 0.28 mg KOH per g.
During each visit, fasting blood was collected, blood pressure was measured and the subjects completed a questionnaire to obtain information about changes in medication, lifestyle habits (e.g. physical activity), and tolerability of linseed oil. The questionnaire additionally included questions about dietary changes during the study. Moreover, the participants were requested to restrict their dietary n3 PUFA intake four weeks before and throughout the study, e.g. abstain from fatty fish (salmon, herring, tuna and mackerel) and ALA-rich vegetable foodstuffs such as linseeds, chia or walnuts (including oils), to minimize the effects on the variability of ALA and LC PUFA intake and blood levels. Prior to visit week 0, week 6, and week 12, the subjects completed a 3-day dietary questionnaire including two working days and one weekend day. The dietary questionnaires were analyzed using PRODI (Nutri-Science GmbH, Freiburg, Germany) to obtain the data on energy and nutrient intake. The total ALA intake was calculated retrospectively by adding the ALA intake from the background diet to the ALA dose from the linseed oil intake.
Blood samples were collected in the morning between 6:30 and 11:00 a.m. after overnight fasting. The examinations were scheduled at the same time for each subject. The samples were obtained by venipuncture of an arm vein using Multiflyneedles (Sarstedt, Nümbrecht, Germany) into serum and EDTA monovettes (Sarstedt). For the analysis of fatty acids in the plasma, EDTA blood monovettes were centrifuged for 10 min at 1500g and 4°C and the plasma was transferred into 1.5 mL plastic tubes (Sarstedt) and immediately frozen and stored at −80°C until extraction and analysis. For the analysis of fatty acids in RBCs, the cell sediment after centrifugation for 10 min at 1500g and 4°C and removal of the plasma was washed twice with PBS (containing 1.5 mg mL −1 EDTA). Finally, the RBCs were reconstituted in PBS to the initial blood volume, transferred into 1.5 mL Eppendorf tubes and immediately frozen and stored at −80°C until extraction and analysis. All transfer steps were carried out on ice. Other sets of blood samples (serum and EDTA monovettes) were sent to external laboratories for the measurement of clinical parameters. Serum lipid levels, liver enzymes and small blood picture were determined in the LADR laboratory (Laborärztliche Arbeitsgemeinschaft für Diagnostik und Rationalisierung e.V.), Hannover, Germany.

Study population
Participants were recruited from the general population in Hannover, Germany through advertisements. Several selection criteria were defined to assemble a homogeneous study collective. In particular, only men within a narrow age range from 20 to 40 years were included to minimize potential fluctuations in lipid profiles due to age or hormonal influence. Subjects were preselected via screening questionnaires according to the following inclusion criteria: male sex, age between 20 and 40 years, body mass index (BMI) between 20 and 27 kg m −2 , and mixed diet with low meat and fish consumption. The exclusion criteria were defined as follows: smoking, serum triglyceride levels ≥150 mg dl −1 (≥1.7 mmol l −1 ), serum total cholesterol levels ≥200 mg dl −1 (≥5.2 mmol l −1 ), a relative amount of ΣEPA + DHA in whole blood ≤3 and ≥6%, intake of fish (>2 times per week) as well as addiction to alcohol, drugs and/or medications and diseases: chronic diseases (e.g. malignant tumors, manifest cardiovascular disease, insulin-dependent type 1 and 2 diabetes, and severe renal or liver diseases); chronic gastrointestinal disorders (especially small intestine, pancreas, and liver) as well as prior gastrointestinal surgical procedures (e.g. gastrectomy); hormonal disorders (e.g. Cushing's syndrome and untreated hyperthyroidism); uncontrolled hypertension; blood coagulation disorders and intake of coagulationinhibiting drugs; periodic intake of laxatives; intake of antiinflammatory drugs (including acetylsalicylic acid); and intake of lipid lowering drugs or supplements during the last 3 months before baseline examination. The inclusion and exclusion criteria were assessed via questionnaires. The pre-selected subjects were invited for a screening examination to collect fasting blood for the analysis of serum lipid levels, liver enzymes and fatty acid profiles in whole blood.

Fatty acid analysis
The concentrations of fatty acids were determined by means of gas chromatography (GC) with flame ionization detection as described 37 with slight modifications. In brief, lipids were extracted with MTBE/MeOH and derivatized with methanolic hydrogen chloride, and the resulting fatty acid methyl esters (FAME) were quantified using methyl pentacosanoate (C25:0 FAME) as the internal standard (IS). In addition to the determined concentration, reported as µg fatty acid per mL blood, the relative amount (% of total fatty acids) of each fatty acid was calculated directly based on peak areas as described. 37

Oxylipin analysis
The concentrations of oxylipins in the plasma were determined by means of an established liquid chromatography-mass spectrometry (LC-MS) based targeted metabolomics platform (Table S2 †) as described [38][39][40] with slight modifications. In brief, after the addition of the IS and antioxidant solution, the plasma samples were diluted with 1 M sodium acetate in water : MeOH (95 : 5 v : v) adjusted to pH 6.0 with acetic acid. Additionally, 10 µL of a solution of the LOX inhibitor 2-(1thienyl)ethyl 3,4-dihydroxybenzylidenecyanoacetate (2 µM) and the protease inhibitor phenylmethylsulfonyl fluoride (5 mM) was added. The extraction was carried out on a nonpolar (C8)/anion exchange mixed mode material (Bond Elut Certify II, 200 mg, Agilent) utilizing ethyl acetate/n-hexane (75 : 25, v : v) with 1% acetic acid as the eluent. The LC-MS measurement was carried out in scheduled selected reaction monitoring following negative electrospray ionization and the quantification of oxylipins was performed by external calibration utilizing 13 deuterated IS. [38][39][40]
The distributions of the sample sets were analyzed by means of the Kolmogorov-Smirnov test. The differences between baseline (week 0) levels and different time points after high-ALA diet (week 1, week 3, week 6, week 12) as well as between week 12 and week 14 and week 20 were analyzed by ANOVA for repeated measurements with the acceptance of statistical significance at p ≤ 0.05. To determine the statistical significance between baseline levels and each time point (as well as between week 12 and week 14 and week 20), t-tests for paired samples were carried out. For t-tests, the significance levels were adjusted according to the Holm-Bonferroni method. All statistical analyses were carried out with the SPSS software (Version 24, SPSS Inc., Chicago, IL, USA).

Study population
Twenty male subjects met the criteria and thus were included in the study. All participants (mean age 26.2 ± 4.53 years) were healthy and had a normal BMI (24.9 ± 2.0 kg m −2 ) and serum lipid profile ( Table 1). The eating habits (especially fish consumption) and the physical activity of the probands did not change during the intervention period as investigated by dietary questionnaires. The study collective consumed a normal mixed diet (including meat) with low fish consumption (≤1 fish serving per week) and low fruit and vegetable consumption (1-2 portions per day) and had a medium physical activity status (3-5 hours of sports per week) and a high education level (advanced technical college certificate). One subject withdrew his consent after 1 week of the intervention. All other 19 participants completed the 12-week intervention period and attended at all five intervention time points. At week 14 of the follow-up period, only 17, and at week 20, 13 participants attended the examinations. Linseed oil was well tolerated and no adverse effects were reported during the intervention period.
The evaluation of the 3-day dietary questionnaires showed that the variability of PUFA intake other than ALA was minimal ( Table 2). For LA, AA, EPA, DPAn3 and DHA intake, no significant changes occurred during the intervention period. The intake of ALA from the background diet was low with minimal variability. The total ALA intake was 1.39 ± 1.31 g d −1 at week 0, 13.9 ± 0.34 g d −1 at week 6 and 14.0 ± 0.53 g d −1 at week 12. Due to the high-ALA diet, the total PUFA intake significantly (p < 0.001) increased from week 0 (11.2 ± 7.02 g d −1 ) to week 6 (23.3 ± 3.14 g d −1 ) and week 12 (22.9 ± 5.87 g d −1 ). The energy and total fat intake did not change significantly; however, the energy intake was lower at week 12 (2403 ± 524 kcal d −1 ) compared to week 0 (2910 ± 1181 kcal d −1 ) and week 6 (2924 ± 820 kcal d −1 ) ( Table 2). In addition, for saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) intake, no significant changes were detected during the intervention period. The intake of SFA decreased (n.s.) from week 0 (31.1 ± 15.5 g d −1 ) to week 6 (26.9 ± 11.0 g d −1 ) and week 12 (24.4 ± 9.83 g d −1 ). Although not significant, the intake of EPA, DPAn3 and DHA slightly decreased during the intervention period (Table 2).
Body weight, BMI, blood pressure and total cholesterol (TC) levels were unchanged during the intervention (Table 1). Also, Table 1 Clinical, biochemical and anthropometric parameters of the study population Week 0 Week 1

t-Test a
Week 3

t-Test a
Week 6

t-Test a
Week 12

0.026
The levels are shown at week 0, 1, 3, 6, and 12 of the high-ALA diet (14.0 ± 0.45 g d −1 ). BMI: body mass index; dias BP: diastolic blood pressure; HDL: high density lipoprotein; LDL: low density lipoprotein; n.s.: not significant; SD: standard deviation; sys BP: systolic blood pressure; TC: total cholesterol; TG: triglycerides, wk: week. a t-Test for paired samples with Holm-Bonferroni correction; significance level p and triglyceride (TG) levels did not change during the intervention period even though statistically significant lower LDL levels (108 ± 18.5 to 96.9 ± 16.6 mg dl −1 ) were detected after 1 week (no significant differences after 3, 6 and 12 weeks).

Changes of fatty acid profile in RBCs
At baseline, the relative amount of ΣEPA + DHA in the RBCs of the study collective was 4.63 ± 0.19% of total fatty acids (Table 3). Thus, all subjects had a low ΣEPA + DHA status within a narrow range. Prior to the intervention, AA was present in the highest concentrations in the RBCs (150 ± 3.90 µg mL −1 ) among all PUFAs, followed by LA (104 ± 3.60 µg mL −1 ), DHA (41.0 ± 1.93 µg mL −1 ), C22:4n6 (31.6 ± 1.01 µg mL −1 ), DPAn3 (25.9 ± 0.82 µg mL −1 ) and EPA (6.13 ± 0.51 µg mL −1 ) ( Table 3). The ALA concentrations in the RBCs were low with 1.44 ± 0.10 µg mL −1 at week 0. In the course of the high-ALA diet, the concentrations and relative amount of ALA, EPA and DPAn3 in the RBCs ( Fig. 1A-C) increased significantly and decreased again in the follow-up period, whereas the DHA concentrations decreased in response to the high-ALA diet (Fig. 1D). In the following, only the concentrations are discussed unless the relative fatty acid distribution showed a different trend. It is noteworthy that the relative amount of ΣEPA + DHA in the RBCs did not change in response to the high-ALA diet ( Table 3).
The initial AA concentrations in the RBCs (150 ± 3.90 µg mL −1 ) were only marginally reduced (136-138 µg mL −1 ) in the first 6 weeks and then significantly ( p = 0.001) dropped to 124 ± 3.53 µg mL −1 at week 12 (Table 3, Fig. 2), corresponding to a mean decrease of 16.6 ± 3.31%. The relative AA amount was statistically significantly reduced at all time points of the intervention. In the follow-up period, the AA concentrations increased, although not significantly, whereas the relative increase was significant ( p = 0.012 (week 14) and p = 0.001 (week 20)) ( Table S3 †).
Both the absolute concentrations and relative amount of LA in the RBCs did not change in the course of the high-ALA diet and follow-up period (Tables 3 and S3 †). The concentration of SFA, PUFA and Σn3 PUFA in the RBCs remained constant during the study. However, the concentrations of MUFA and Σn6 PUFA in the RBCs were significantly decreased after 12 weeks of the high-ALA diet ( Table 3). The MUFA concentrations decreased ( p = 0.019) from 210 ± 6.24 µg mL −1 at week 0 to 179 ± 5.89 µg mL −1 at week 12, while the Σn6 PUFA con-     centrations decreased ( p = 0.004) from 303 ± 6.55 µg mL −1 at week 0 to 261 ± 8.01 µg mL −1 at week 12 ( Table 3). The ratio of Σn6 to Σn3 PUFA significantly declined ( p < 0.001) after 1 week of the high-ALA diet from 4.15 ± 0.13 (week 0) to 3.81 ± 0.10 (week 1) and further to 3.30 ± 0.07 after week 12 (Table 3). In the follow-up period, the Σn6/Σn3 PUFA ratio increased ( p = 0.002) again to 3.73 ± 0.13 (week 20) (Table S3 †).
LA-derived oxylipins remained unchanged; however, the concentrations varied in the course of the trial (Fig. 2, Table S4 †). This is consistent with the LA concentration in the RBCs that did not show significant changes in response to the high-ALA diet.
For most of the AA-derived hydroxy-and dihydroxy-PUFA, such as 5-, 15-, 20-HETE and 14,15-DiHETrE, a slight though statistically not significant decrease in concentration after 1 week of ALA supplementation was observed (Fig. 2). Similarly, no consistent shift of DHA-derived oxylipins was observed (Fig. 2, Table S4 †). Overall, the oxylipin levels were reflected by the respective precursor fatty acid concentrations in the RBCs, though changes in the oxylipin levels occurred at later time points and were, due to higher SE, less pronounced compared to the precursor fatty acids.

Discussion
Factors like age, 43,44 BMI, 44 smoking 45 and genotype 46 showed an influence on the LC n3 PUFA status, especially on the conversion from ALA to DHA. Recently, it has been shown that the activity of endogenous EPA and DHA synthesis adapted during evolution in the presence of these fatty acids in the diet. 47 Several studies observed higher circulating relative DHA amounts in women compared to men independent of dietary intake 48-50 and higher conversion rates from ALA and EPA to DHA 51 possibly due to the influence of estrogen on the PUFA metabolism. 52 Consequently, to minimize the variability, a homogeneous collective of healthy, non-smoking men within a narrow range regarding age (mean age 26.2 ± 4.53 years) and BMI (24.9 ± 2.0 kg m −2 ) was chosen to investigate the effect of ALA on the LC n3-PUFA levels.
In addition, to study the effect of a high daily ALA intake in a Western diet on the EPA and DHA blood levels, it is crucial to choose a study collective with low baseline EPA/DHA levels, since the expected conversion of ALA to EPA and DHA is the highest compared to subjects with a moderate or high EPA/ DHA status.
We selected probands basically eating a mixed Western diet with a low meat and fish consumption and explicitly screened for low blood LC n3 PUFA status. The relative ΣEPA + DHA level in RBCs, similar to omega-3 index, was 4.15 ± 0.13% of total fatty acids and comparable to the (low) mean omega-3 index of men in the U.S. and Germany which is associated with the risk of cardiovascular disease. 53 As expected, the high-ALA dietproviding an ALA amount of about 4.7 en%resulted in a strong increase in the ALA levels in the RBCs. The incorporation of ALA (and other PUFAs) into the RBCs is determined by the blood cell turnover (the mean life span of a red blood cell is approximately 120 days in circulation) and thus the ALA and its bioconverted longer chain n3 PUFAs EPA and DHA do not fully reach the RBCs. Nevertheless, a 238 ± 24% increase of ALA concentration in the RBCs was observed after one week.
The ratio of LA to ALA in the RBCs (74.8 ± 3.54) at baseline is much higher than expected from the dietary supply of these C18 PUFAs (LA/ALA intake ratio: 7.70 ± 3.75). Even after a 12-week intake of similarly high amounts of LA (9.38 ± 3.08 g d −1 ) and ALA (14.0 ± 0.45 g d −1 ) with a ratio of 0.67 ± 0.21, the ratio of LA to ALA in the RBCs remained high (17.5 ± 0.97). The possible reasons for this could be that ALA significantly differs from LA in absorption, tissue distribution, membrane incorporation, and/or degradation. Most likely, a high percentage of 60-85% of ALA is rapidly degraded by beta-oxidation 54 before it becomes available for tissue distribution and membrane integration as well as elongation and desaturation to EPA.
The high-ALA diet also affected the concentrations of other PUFAs with significantly increased EPA and DPAn3 as well as significantly decreased DHA and AA concentrations in the RBCs. Of note, the concentration of EPA was with a change of about 5 µg mL −1 similarly elevated as the ALA concentration. The percentage increase in the concentrations and relative amounts was more pronounced for EPA (week 12: 97 ± 21% and 112 ± 17%, respectively) compared to DPAn3 (week 12: 27 ± 7% and 39 ± 4%, respectively). It is likely that increasing EPA levels are the result of a conversion from ALA; however, it cannot be excluded that the retroconversion of DHA to DPAn3 and EPA occurred, which would also ( partly) explain decreasing DHA concentrations. These results are consistent with previous studies, where a dose-dependent and higher increase of EPA compared to DPAn3 was observed in the RBCs 55,56 and plasma and platelet phospholipids. 35 However, supplementing EPA is more efficacious compared to ALA in raising the EPA blood levels. 19 A six-week supplementation of 600 mg EPA per day, which corresponds to a ∼10-fold increase compared to the median intake in Germany, resulted in a 138% increase of the relative serum phospholipid EPA amount. 57 A higher dose of 2.0 g EPA per day caused a 325% increase of the relative EPA amount in the RBCs. 58 The initial ALA concentrations were also low (1.44 ± 0.10 µg mL −1 ) compared to the EPA concentrations (6.13 ± 0.51 µg mL −1 ) and the ratio of ALA to EPA increased in the first three weeks and slightly decreased thereafter. The high-ALA diet led to a strong increase of ALA in the RBCs. However, as discussed before, with increasing ALA concentrations, the rate of ALA catabolism increases 24 as observed in the moderate drop of the ALA levels between week 6 and week 12. This may also explain the steady state in the EPA concentrations between week 6 (10.9 ± 0.67 µg mL −1 ) and week 12 (11.0 ± 0.64 µg mL −1 ).
The observed reduction of the DHA levels is surprising and in contrast to earlier studies, which determined a conversion (rate) of ALA to DHA of 0.5-5%. [16][17][18][19][20][21] However, in these studies, the ingested ALA amount was low (1.0-3.5 g) 17,59 compared to ours. A study with a similar design as our study including a high daily ALA dose (15.4 ± 7.5 g) showed no significant effect on relative DHA amounts in plasma and platelet phospholipids, 35 possibly due to low sample size (n = 7), an inhomogeneous initial LC n3 PUFA status and a highly variable ALA intake (high SD). In contrast, we observed significantly decreased DHA concentrations and relative amounts in the RBCs in response to the high-ALA diet. It should be noted that the biological variability of fatty acid concentrations in the RBCs is small compared to plasma and plasma phospholipids. 60 Nonetheless, several studies indicated a tendency for declining DHA levels in response to high ALA consumption, which was in most cases marginal and not statistically significant. 35,[61][62][63][64][65][66][67][68][69] One possible explanation for the decreasing DHA concentrations in the RBCs may be that DHA accumulates in the nerve cells of the brain. 70 While human studies are limited to blood as the medium of investigation, animal studies demonstrated that the conversion of ALA to EPA and DHA is tissue specific. In a rat study with high-ALA chia seed supplementation, the accumulation of DHA in the heart and liver was observed, while the plasma DHA concentrations remained constant. 71 In our study, the ALA intake from the background diet was tightly controlled and hence the variability of total daily ALA intake in the intervention period was low (∼1.9 g). Only a few other studies found a significant reduction of the relative DHA levels in mononuclear cell phospholipids 66 69 was much higher than in our study (<10 g) based on food questionnaires ( Table 2).
The conversion efficiency of ALA to DHA appears to be affected by a high LA, ALA and total PUFA intake. A rat study observed the highest conversion of ALA to DHA as a result of feeding a narrow dietary range of 1-2 en% LA and 1-3 en% ALA, while the DHA levels were suppressed to basal levels (∼2% total fatty acids) with the total PUFA levels above 3 en%. 72 Excessive LA and ALA compete with LC n3 PUFAs for the rate-limiting enzyme Δ6-desaturase. 73 Δ6-desaturase catalyzes the desaturation of LA to GLA, of ALA to stearidonic acid (C18:4n3) and also of tetracosapentaenoic acid (C24:5n3) to tetracosahexaenoic acid (C24:6n3), which is finally shortened to form DHA by peroxisomal β-oxidation. 15 The intake of LA, AA, EPA, DPAn3, and DHA did not significantly change during the intervention compared to baseline (3-day dietary questionnaires Table 2). In addition, the ALA intake from the background diet (minus the ALA intake via the daily linseed oil ingestion) did not change between week 0, week 6 and week 12. On the other hand, the intake of EPA, DPAn3, and DHA via the background diet was slightly decreased, possibly due to the advice to avoid oily fish meals during the intervention time. However, it seems unlikely that this statistically insignificant decline caused the observed decrease in the DHA concentrations in the RBCs.
However, it should be noted that estimates of dietary fat intake relied on self-reported data and are potentially biased by food choice, incomplete dietary protocols and methodological limitations associated with accurate fatty acid composition data in food databases. 74 The beneficial health effects of LC n3 PUFA are believed to be ( partly) mediated by oxidized mediators formed in the AA cascade. 10 A correlation between higher levels of precursor n3 PUFAs (e.g. EPA and DHA) and their oxylipins was demonstrated in different intervention studies. [75][76][77][78][79] Accordingly, in the present study, the changes of the oxylipin levels in the plasma are generally reflected by the changes of the respective precursor fatty acids in the RBCs. As expected, the levels of ALA-derived oxylipins increased in response to the higher dietary intake of ALA. In contrast to the ALA concentrations in the RBCs, which increased more than 3-fold after only 1 week of ALA supplementation, ALA-derived oxylipins were only slightly but not significantly elevated. Moreover, whilst the ALA concentration in the RBCs seemed to reach a steady state after 6 weeks of the high-ALA diet, ALA-derived oxylipins, such as hydroxy-PUFA 9-and 13-HOTrE, increased steadily until week 12 up to 1.9-fold and 2.6-fold, respectively. The slower and less pronounced rise of ALA-derived oxylipins compared to their precursor fatty acid was not observed for DHA-derived oxylipins compared to the blood cell concentrations of DHA in a similar study. 78,79 A possible explanation is the lower baseline concentration of ALA compared to DHA, which might have led to a more rapid increase and the higher supplemented dose. 77 With regard to oxylipin formation, ALA is mostly discussed as a precursor of LC n3 PUFA; 80 therefore, the biological role of ALA-derived oxylipins is only poorly understood. Some studies demonstrated positive biological effects of ALA-derived oxylipins; 81,82 however, further investigation of these mediators needs to be carried out as ALA-derived oxylipins are present in relevant concentrations in humans on a Western diet. 10 Consistent with the elevated levels of EPA in the RBCs (1.8fold increase at week 6), a high-ALA diet leads to increasing concentrations of EPA-derived hydroxy-and dihydroxy-PUFA in the plasma (∼1.3-to 1.8-fold at week 6). The higher levels of EPA-derived oxylipins upon a high-ALA diet may have beneficial health effects, e.g. 18-HEPEa precursor of pro-resolving and anti-inflammatory E-series resolvins 83concentrations increased 1.7-fold at week 6. However, it has to be noted that direct supplementation with EPA raises the EPAderived oxylipin levels more efficiently. 84 Although the DHA concentrations in the RBCs were significantly lowered in response to the high-ALA diet, the DHAderived oxylipin levels showed no consistent shift towards lower levels.

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The conversion rates of ALA to EPA and DHA as well as the formation of oxylipins from n3 PUFA are influenced by the presence of n6 PUFA competing for the same enzymes. 15,85 Several studies demonstrated that n3 PUFA supplementation leads to declining AA and AA-derived oxylipin concentration; however, the results were heterogeneous between different intervention studies. 84 Despite the significant decrease of AA in the RBCs in the present study, only a slight but not significant decline of AA-derived oxylipins, e.g. hydroxy-PUFA 5-and 20-HETE, was observed, while no effect was observed for, e.g., the 15-LOX product 15-HETE.
Similar results were obtained for LA-derived oxylipins and no relevant reduction in response to high-ALA intake was observed. Most likely, the excess of LA in the diet (and consequently in the RBC membrane concentration) was too high to be modified by a high-ALA diet. A decrease of the LA/ALA ratio (from 74.8 ± 3.54 (week 0) to 17.5 ± 0.97 (week 12)) and the ratio of LOX-derived hydroxy-PUFA 9-HODE/9-HOTrE (19.4 ± 1.04 (week 0) to 10.6 ± 0.68 (week 12)) and 13-HODE/13-HOTrE (from 20.5 ± 1.39 (week 0) to 7.73 ± 0.78 (week 12)) results from elevated ALA and ALA-derived oxylipin concentrations with constant LA and LA-derived oxylipin concentrations. A reduction of LA and its oxylipins is assumed to be beneficial, as negative health effects were observed for LA metabolites such as sEH products of the CYP-derived epoxy-PUFA. [86][87][88] A reduction of the LA metabolite 9,10-DiHOME by supplementation with a lower (6 g d −1 ) dose of ALA compared to our study was demonstrated by Caligiuri et al. in young individuals (19-28 years), 89 after only 4 weeks of the intervention. However, the participants had to abstain from dietary oils, which might have altered their normal eating habits, thus, leading to shifts in the fatty acid and oxylipin pattern.