The role of dietary n3- and n6- long chain polyunsaturated fatty acids (LC-PUFAS) in human health and disease has been studied for decades. It is generally accepted that PUFAS are involved in inflammatory processes, while n3-PUFAS are rather anti-inflammatory, n6-PUFAS exhibit rather pro-inflammatory activities (1). The World Health Organization (WHO) recommends a dietary intake of PUFAS in a n6/n3-ratio of approximately 4:1, however, the level consumed in a typical Western diet is much higher (2, 3). This disproportion may be partly responsible for the increasing incidence of chronic inflammatory diseases such as cardiovascular diseases (4, 5) or rheumatoid arthritis (6, 7). In good accordance, higher intakes of dietary n3-LC-PUFAS are associated with lower risk for cardiovascular diseases (8, 9).

The most important n3-LC-PUFAS are eicosapentaenoic acid (EPA, C20:5n3) and docosahexaenoic acid (DHA, C22:6n3), which mainly occur in fatty fish, such as mackerel (Scomber scombrus), herring (Clupea harengus), tuna (Thunnus thynnus) and salmon (Salmo salar) (10). However, as marine fish resources are decreasing and the global population is increasing, renewable, plant-derived n3-LC-PUFA-rich alternatives are needed to provide adequate sources of these health-relevant fatty acids. An alternative approach is to obtain EPA- and DHA from their precursors through endogenous synthesis rather than directly through food. Since the endogenous synthesis of n3-LC-PUFA (Figure 1) is strongly affected by substrate levels and the activities of rate-limiting enzymes (11), adding precursor fatty acids to food along with molecules that stimulate their enzymatic conversion could be an important strategy to improve EPA- and DHA levels in humans. Notably, potential precursor fatty acids for EPA and DHA synthesis are found in many plant-derived oils. Flaxseed oil from Linum usitatissimum, for example, consists of ~60% α-linolenic acid (ALA, 18:3n3). Other seed plant oils, such as echium oil (Echium plantagineum), lappula oil (Lappula squarrosa) or ahiflower oil (Buglossoides arvensis) are rich in both, ALA and stearidonic acid (SDA, C18:4n3) comprising 30–40% and 10–20% of the total fatty acids, respectively (12, 13). In theory, SDA bypasses the first ∆6-desaturase (FADS2)-driven desaturation step (Figure 1), which has been considered as rate-limiting within the LC-PUFA synthesis cascade (16–18). In addition, it has been shown in humans that circulating and cellular EPA levels are increased more substantially by ALA- and SDA-rich ahiflower oil than by flaxseed oil that contains only ALA (13). The stimulation of n3-LC-PUFA synthesis by plant bioactives has been studied in cultivated cells in vitro (14, 19), and in vivo in fish [Schiller (20, 21)], chickens (22) and humans (23).

Oxygenation of n3- and n6-PUFAs results in bioactive oxylipins which are known for their anti- or pro-inflammatory mode of action, respectively (24). Enzymatic routes for oxylipin synthesis are catalyzed by lipoxygenases (LOX), cyclooxygenases (COX), cytochrome P450 monooxygenases (CYP450) and soluble epoxid hydrolase (sEH) while resulting metabolites are mainly hydroxy-, epoxy- and dihydroxy-PUFAS (25, 26). Oxylipins can be esterified with phospholipids in membranes or are non-esterified (27). Well known oxylipins such as prostaglandins, thromboxanes and leucotrienes are classical eicosanoids formed from C20 fatty acids (i.e., ARA) which exhibit a wide variety of biological activities (28, 29). For several n3-PUFA oxylipins, such as EPA derived 15-HEPE, anti-inflammatory properties have been described (30). However, the formation, signaling and occurrence and thus the biological roles of the so-called specialized lipid mediators (SPM) or lipoxins are rather unclear (31).

In marine food sources, high EPA and DHA levels are usually accompanied by high amounts of taurine, an amino sulfonic acid (32). Furthermore, taurine has the potential to influence lipid adsorption and metabolism, either as a constituent of bile salts (33, 34) or as a protector of susceptible LC-PUFAs against lipid peroxidation (35–37). The present study adopts a novel approach to investigate whether taurine, either alone or in combination with n3-LC-PUFA precursors, serves as a modulator of the fatty acid pattern in humans and cultivated cells. In humans, the precursor fatty acids and taurine were orally supplied using ahiflower oil and standardized taurine capsules. In parallel, cultivated hepatocytes were treated with isolated ALA and SDA as precursors and EPA and DHA as positive controls. HepG2 cells were chosen for this purpose due to their human origin. Furthermore, HepG2 hepatocytes express desaturases and elongases necessary for the conversion of ALA and SDA to EPA and DHA (38, 39). Previous studies conducted by our group have demonstrated the feasibility of modifying the fatty acid profile through the supplementation of fatty acids and bioactives in the culture media (19). However, to the best of our knowledge, it has not yet been demonstrated experimentally whether equimolar ALA and SDA treatments of cultivated hepatocytes result in differential cellular EPA and DHA accumulation. Furthermore, there is no existing literature data regarding the impact of SDA or ahiflower oil on cellular and circulating oxylipins. The alterations in the fatty acid pattern induced by both in vivo and in vitro treatments prompted an investigation into whether these changes were similarly reflected in an altered oxylipin pattern. Overall, the present study provides novel insights into the capacity of plant derived n3-PUFAS to modulate fatty acid and oxylipin patterns in humans and cultivated cells.

The present human intervention study clearly reveals the ability of dietary ALA and SDA-rich ahiflower oil to improve EPA status in vivo. Circulating EPA levels increased nearly 3-fold, while DHA levels remained largely unaffected due to the ahiflower oil intervention. It is known that in humans, plasma EPA levels are usually lower than DHA levels and respond better to dietary interventions than DHA, although there are large interindividual differences (45). The daily consumption dose of 9 g ahiflower oil accounts for an ALA and SDA intake of ~4 g/d and of ~1.7 g/d, respectively. These intake rates are thus considerably higher than those in human intervention studies involving EPA and DHA-rich oils. For example, 12 weeks of n3-PUFA supplementation with 1.4 g EPA and 0.2 g DHA per day in youth (adolescents) with mood disorders elevated EPA plasma levels nearly 4-fold, while DHA levels were only slightly affected (46). In a bioavailability study involving healthy adults, 4 weeks of daily supplementation with 816 mg of EPA and 522 mg of DHA resulted in approximately a 5-fold increase in EPA plasma levels and a 1.5-fold increase in DHA levels. This effect was observed irrespective of whether EPA and DHA were provided in the form of fish oil ethyl ester, fish oil triglycerides, or krill oil (47). In our study, the conversion rate of ALA and/or SDA to EPA was relatively high, which corresponds with other human studies involving plant-derived ahiflower (13, 48) or echium oil (49). As suggested by these results, compared to ALA-rich linseed oil, ALA and SDA-rich ahiflower (13) and echium oil (49) more greatly increase circulating and cellular EPA levels. With regard to the unaffected DHA levels, a recent study in mice, utilizing C13-labeled fatty acids derived from fish DHA, ahiflower oil and flaxseed oil, also demonstrated that even when DHA levels in serum, liver, adipose tissue, and the brain are not affected by all of the interventions, the synthesis/turnover of DHA increases over time. Moreover, diets rich in DHA and ahiflower oil exhibit similar DHA turnover rates in the liver, adipose, and brain tissues, while turnover rates from flaxseed oil were significantly slower compared to the DHA diet (50). Thus, the study suggests that measuring DHA levels may not provide as accurate an assessment of the potency of precursors for DHA synthesis as determining the synthesis/turnover rate from these precursors.

To examine the capacity of individual n3-PUFAS as precursors for EPA synthesis, we conducted additional mechanistic studies with cultured HepG2 hepatocytes. A 24 h treatment with different n3 fatty acids supplied at equal concentrations significantly increased EPA cellular concentrations, while the extent of final accumulation in % of total FAMEs increased as follows: SDA (1.7) < ALA (5.7) < EPA (9.2) < DHA (16.6). Furthermore, supplementation with ALA and SDA increased the cellular EPA concentration from zero to 3.0 and 5.1% of the total FAMEs, respectively. Under the conditions investigated, the results confirm that SDA was 65% more efficient in enhancing cellular EPA levels than ALA.

When taurine was tested as a putative modulator for the conversion of ALA/SDA to EPA/DHA in vivo, we found that the circulating fatty acid pattern was not affected regardless if taurine was consumed alone or in combination with ahiflower oil. Likewise, taurine treatment did not alter the n3-PUFA levels of control, ALA or SDA-supplemented HepG2 cells. Hence, our data suggest that although taurine is present in n3-PUFA-rich tissues at high concentrations such as the retina (51), it does not impact PUFA accumulation or synthesis. However, the association of PUFAS and taurine may protect PUFAS against lipid peroxidation (35–37) and support osmoregulatory functions (52). In mice, dietary EPA and DHA provision (wild-type C57BL/6 mice) increased the levels of EPA and DHA more efficiently than improved endogenous de novo synthesis in transgenic fat-1 mice (53). Moreover, by employing this mouse model, a recent study showed better protection from high-fat diet induced metabolic and hepatic disorders was obtained with endogenously generated n3-PUFAs than dietary n3-PUFAs (54). Therefore, the level and origin of EPA and DHA (endogenous vs. exogenous) should be investigated.

The main synthesis routes for n3 EPA and n6 ARA utilize the same desaturases and elongases (Figure 1). Apart from its high content of n3-PUFAs, ahiflower oil also contains significant amounts of n6 linoleic acid (LA) (9–15%) which is the precursor for ARA synthesis. Interestingly, LA intake via ahiflower oil did not lead to an increase of ARA levels in human plasma but a slight yet nonsignificant decrease. In theory, the conversion of ALA and LA to form EPA and ARA compete for the same desaturases and elongases. Enhanced EPA synthesis might come at the expense of ARA synthesis. However, this observation has not been reported by others who supplemented humans with plant derived oil rich in ALA (55) and/or SDA (13, 49).

In cultivated HepG2 cells, only ALA and DHA treatment significantly decreased the cellular ARA content. However, cellular fatty acid levels were expressed in % of total FAMEs, which shift with additional accumulation of supplemented ALA and DHA. Therefore, the effects on ARA observed in humans and HepG2 cells may be negligible. The cell culture results also reveal an alternative pathway from ALA to EPA, as ALA-treated cells accumulate its elongation product ETE, but neither SDA nor ETA were found in these samples. Hence, in this cell model ALA is initially elongated to ETE by the action of ELOVL5 and is not desaturated to SDA by the Δ6-FADS2-catalyzed reaction. The conversion of radio-labeled ALA to ETE has already been achieved in cultured hepatoma 7288 C cells (56–58). Furthermore, in vivo studies with salmon (59) and zebrafish (60) revealed that partial knockout of the fads2 gene resulted in the accumulation of LA and ALA C20-elongation products, which is indicative of alternative elongation prior to Δ8-FADS2-driven desaturation. Another explanation for the absence of SDA accumulation in ALA-treated cells was given by Park et al. (61), who described that SDA undergoes a coupled elongation immediately after Δ6-FADS2-driven desaturation, concomitantly restraining cellular SDA accumulation (61). In accordance with results from the current human intervention study, neither ALA nor SDA significantly increased cellular DHA levels in cultured HepG2 cells. In line with previous studies on fatty acid pattern in cultured hepatoma cell lines (19, 62), DHA was found to be the most abundant n3-PUFA in both RPMI and the vehicle control (EtOH) cells. According to Else et al. (63), FBS contains considerable amounts of DHA (~ 2–3% of total fatty acids) and in many cases also its precursor fatty acids ALA, SDA, EPA and DPA. Therefore, we hypothesize that the cellular accumulation of DHA supplied by FBS in the media and baseline synthesis of DHA from its precursors is already substantial, making further modulation challenging.

Both n3- and n6-PUFAS are precursors of bioactive eicosanoids and other oxylipins (1). Accordingly, we found that in humans, oral intake of ahiflower oil, which predominantly led to elevated plasma levels of EPA, significantly increased the concentration of EPA-derived eicosanoids in plasma, in contrast, DHA-derived oxylipins were less affected (only 14-HDHA and 16-HDHA levels were slightly decreased). This supports the assumption that the formation of oxylipins is partly determined by the content of their respective precursor fatty acids. Similar outcomes have been observed in previous feeding studies, in which dietary intake of EPA and DHA increased the circulating amounts of the respective oxylipins in mice (64) and dose-dependently in humans (26). However, this may not apply to ALA in our results, since supplementation with ahiflower oil significantly increased the plasma levels of ALA, without affecting the plasma levels of ALA-derived non-esterified oxylipins. A change in erythrocyte ALA and plasma ALA-derived oxylipins was observed in humans receiving ALA-rich linseed oil in a 12-week intervention (65). However, the ALA dose (12.9 g/d) was much higher than that offered to the participants within the current study (4 g/d). Moreover, in a study by Greupner et al. (65), ALA levels increased after 1 week, while the highest levels of ALA-derived oxylipins were observed after 6 weeks of intervention (65). These results reveal that the oxylipin response was delayed and that the intervention duration of the current study (20 days) was probably too short for ALA oxylipin to be found. In this context, it is worth mentioning a study with obese women, in which the plasma oxylipin pattern was modified by oral supplementation with DHA via fish oil but not ALA-rich flaxseed oil (66). Remarkably, experimental conditions, such as duration of the intervention (28 days vs. 20 days), amount of ALA intake (4 g/day) and sample type (plasma), were similar to those in the current study. In the study by Pauls et al. (66) the plasma levels of EPA and EPA-derived oxylipins were not affected by flaxseed oil intake, which further reveals that EPA-derived oxylipins correspond with plasma EPA levels. In addition, compared to ALA derived from flaxseed oil, SDA derived from ahiflower oil triggers the formation of EPA and EPA-oxylipins more efficiently. It has previously been shown that increasing n3-PUFA status by EPA and DHA supplementation in turn decreases the levels of ARA in erythrocytes and ARA-derived epoxy (CYP450 route) as well as dihydroxy (CYP450/sEH route) oxylipins in human serum (67). Within the current intervention study, ARA and ARA-derived epoxy as well as dihydroxy metabolites were not affected by the ahiflower oil ingestion. Instead, only the levels of 12-HETE and 15-HETE slightly decreased, while EPA counterparts 5-HEPE, 12-HEPE and 15-HEPE increased in response to the ahiflower oil intervention. Furthermore, dihydroxy-EPA metabolites (8,9-DiHETE, 11,12-DiHETE, 14,15-DiHETE, 17,18-DIHETE) were significantly higher following ahiflower oil supplementation, while epoxy-EPA metabolites were not detectable in all plasma samples. Consequently, plant-derived ahiflower oil efficiently increases plasma EPA and corresponding eicosanoids and causes a distinct shift in the entire oxylipin pattern (67).

Fatty acids compete for the enzymes LOX, COX and CYP450. Within the current study cells treated with ALA and SDA contained higher levels of hydroxy ALA, EPA, and DHA metabolites derived from the LOX pathway but hydroxy LA and ARA metabolites were not significantly affected compared to control cells. Moreover, compared to control cells, ALA and SDA supplemented cells contained considerably higher amounts of epoxy-ALA and epoxy-EPA oxylipins. Epoxy-PUFAs can be formed by CYP450 monooxygenases-catalyzed reactions (68). Overall, the present study provides substantial evidence that SDA is a more powerful modulator of EPA and DHA oxylipin levels than ALA. Further investigations are needed to determine if and to what extent SDA acts by enhancing substrate availability (especially EPA) for oxygenation or by stimulating enzyme activity.

In conclusion, ALA and SDA-rich ahiflower oil could serve as an alternative plant-derived source to increase EPA levels in normal weight, healthy humans, while changes in DHA levels are not expected. The increase in plasma EPA levels via ahiflower oil ingestion concomitantly increases EPA-derived hydroxy- and dihydroxy-oxylipins. Additional cell culture results revealed that ALA and SDA increases EPA- levels and EPA-derived oxylipins in HepG2 cells, whereby SDA is more potent than ALA. Taurine does not modulate fatty acid patterns in humans or cultivated HepG2 cells.

The human intervention study was conducted in a crossover design that accounts for interindividual variability effects. Ahiflower oil and taurine were administered as capsules, which improved the oxidative stability of susceptible n3 fatty acids. Furthermore, the capsules could be stored more easily, daily intake procedures were simpler and the compliance of the participants increased.

The results are limited because the study involved a very homogenous group of participants (young healthy men with normal weight). The duration of the intervention phases was relatively short and could be prolonged to further analyze fatty acids/oxylipins in blood cells (e.g., peripheral blood mononuclear cells, erythrocytes) or clinical biomarkers. The HepG2 cell model was utilized to investigate the impact of supplemented n3-PUFAs on cellular fatty acid profiles, particularly focusing on the primary targets EPA and DHA. Given that isolated fatty acids were provided in high supraphysiological concentrations (50 μmol/L), it cannot be excluded that some fatty acids may remain in the medium, and that cellular uptake and accumulation may vary among fatty acids. Taurine was administered at a concentration of 1 mmol/L, a level considered physiological. Nevertheless, concentration-dependent effects should be anticipated in future investigations.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

The studies involving humans were approved by Ethics committee of the medical faculty, Christian-Albrechts-University Kiel, Germany (D 484/18). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

US: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. KE: Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – review & editing. MW: Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – review & editing. KL: Writing – original draft, Writing – review & editing. II: Conceptualization, Data curation, Formal analysis, Validation, Writing – review & editing. FH: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – review & editing. PW: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing. AB-W: Conceptualization, Methodology, Project administration, Supervision, Writing – review & editing. NS: Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – review & editing. GR: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing.