The study was reviewed and approved by the East of England-Cambridge Central Research Ethics Committee (approval number 19/EE/0096), and all participants gave written informed consent.

Inclusion criteria for the study were 18–30 years, body mass index 18.5 and 30.0 kg/m², systolic blood pressure ≤140 mm/Hg, diastolic blood pressure ≤90 mm/Hg, random total cholesterol concentration <7.5 mmol/L, HbA1c concentration <42 mmol/mol (or <6%), C-reactive protein (CRP) concentration <3 mg/L, not consuming fish oil or other oil or dietary supplements, non-smoking, absence of chronic disease, willingness to adhere to the study protocol, and being able to provide written informed consent. Volunteers were excluded from the study if they did not meet the inclusion criteria, were pregnant or planning to become pregnant within the study period, or were participating in another clinical trial. Non-fasting venous blood samples (100 ml) were collected into vials containing lithium heparin anticoagulant on three occasions separated by an interval of 4 weeks. Participants were 10 healthy women aged 26.2 ± 0.8 years with body mass index 23.1 ± 0.4 kg/m² and blood pressure (systolic 106 ± 3 mmHg; diastolic 62.7 ± 2 mmHg), total plasma cholesterol (4.0 ± 0.2 mmol/l), CRP (1.1 ± 0.1 mg/L), and HbA1c (30.5 ± 1.0 mmol/L) concentrations within normal ranges.

Whole blood was layered onto a histopaque density cushion, and erythrocytes and granulocytes were removed by centrifugation at 845 × g for 15 min at room temperature. PBMCs were collected by aspiration and diluted 1:1 with RPMI1640 containing 10% (v/v) autologous pooled heat-inactivated serum. CD3⁺ T cells were isolated by negative selection using the T-cell EasySep kit (StemCell Technologies) as instructed by the manufacturer. Isolated T cells were washed with 10 ml of RPMI1640 containing 10% (v/v) autologous pooled heat-inactivated serum and collected by centrifugation at 300 × g for 10 min at room temperature. Cryopreservation was carried out as described (38, 39). Ice-cold RPMI1640 medium containing 20% (v/v) dimethylsulphoxide and 10% fetal bovine serum was added to the T-cell pellet, and the cells were frozen at -80°C overnight and then transferred to liquid nitrogen for storage until used.

T-cell culture was carried out essentially as described (34). Briefly, cryopreserved cells were thawed and resuspended in RPMI1640 containing 2 mM of L-glutamine, 100 units/ml of penicillin, and 100 µg/ml of streptomycin and 10% (v/v) heat-inactivated pooled human serum (Sigma-Aldrich) ( Supplementary Table S1 ) and adjusted to a density of 1 × 10⁶ cells/ml. The fatty acid composition of the medium was standardised by using pooled homologous serum instead of autologous serum. The fatty acid composition of the medium was adjusted by addition of 18:2n-6 or 18:3n-3 as free fatty acids to a final ratio of either 8:1 or 5:1 (EFA ratio; Table 1 ). Total 18:2n-6 and 18:3n-3 concentrations included ethyl-[d₅]18:2n-6 (2 µmol/L) and [1-¹³C]18:3n-3 (2 or 4 µmol/L, according to the EFA ratio 8:1 or 5:1, respectively). In order to test whether 18:2n-6 metabolism was altered by the presence of an esterified ethyl group, uptake and conversion of ethyl-[d₅]18:2n-6 (2 µmol/L) were compared to those of unesterified [d₅]18:2n-6 (2 µmol/L) in cultures with an EFA ratio of 5:1 (n = 5 per molecular form with or without mitogen stimulation). Cultures were placed in a humidified incubator in an atmosphere containing 5% (v/v) CO₂ for up to 48 h with or without concanavalin A (Con A; 10 μg/ml; Sigma-Aldrich). Cells were collected by centrifugation and washed as before, and then either snap-frozen and stored at −80°C or processed immediately by flow cytometry.

T-cell activation was assessed by the cell surface expression of CD69 as described (34, 40). Briefly, cells were incubated with PE-Cy7-conjugated anti-human CD69 monoclonal antibody (catalogue number 557745, BD Biosciences) for 30 min at 4°C in the dark, processed for flow cytometry, and analysed using a FACSCalibur (B&D Biosciences) flow cytometer as described (34).

Culture medium (0.9 ml) was thawed, and purified T cells were thawed and suspended in 0.9% (v/v) NaCl. Then, 17:0 (3 µg) was added as internal standard to purified T cells, and 10 µg of 17:0 internal standard was added to culture medium samples. Total lipids were extracted with chloroform/methanol (2:1, v/v) (41). Fatty acid methyl esters (FAMEs) were synthesised from total cell and culture medium lipids by incubation with methanol containing 2% (v/v) sulphuric acid at 50°C for 2 h (42). The reaction mixture was cooled to room temperature and neutralised with a solution of KHCO₃ (0.25 M) and K₂CO₃ (0.5 M). FAMEs were collected by extraction with hexane (42).

FAMEs were resolved on a BPX-70 fused silica capillary column (30 m × 0.25 mm × 25 μm) fitted in an Agilent 6890 gas chromatograph equipped with flame ionisation detection (GC-FID) (43). FAMEs (1 μl) were injected in split mode at an inlet temperature of 300°C with helium carrier at a flow rate of 1 ml/min (44). The oven temperature was held at 115°C for 2 min post-injection, then increased at 10°C/min to 200°C and held for 16 min. The oven temperature was then increased at 60°C/min to 240°C and held for 2 min. The detector was maintained at 300°C. Chromatograms were integrated manually using ChemStation software (version B.03.01, Agilent Technologies), and the amount of cellular fatty acids, expressed as nmol/10⁶ cells, was calculated by comparison of the peak area of each fatty acid of interest to that of the internal standard and adjusted for the number of cells that were extracted. Fatty acid concentrations in culture media were calculated by comparison of the peak area of each fatty acid of interest to that of the internal standard and adjusted for the volume of media that was extracted. Fatty acids were identified by their retention times relative to standards (37 FAMEs, Sigma-Aldrich).

The purity of FAMEs from T-cell extractions was tested by gas chromatography (GC)–mass spectrometry using a mass scan m/z 50–550. Samples were reconstituted in 50 µl of hexane, and a 1-µl injection volume in splitless mode was used with a column flow of 1.5 ml/min. FAMEs were resolved on a Supelcowax 10 capillary column (30 m × 0.25 mm × 0.25 μm film thickness; Supelco) on a 6890 gas chromatograph (Agilent, UK) equipped with a mass selective detector (Agilent 5975). The inlet and detector were set to 250°C, and helium was used as carrier gas. The temperature gradient was set to start at 60°C and held for 3 min, then raised at 12°C/min to 200°C and held for 6 min, followed by a second increase at 12°C/min to 240°C and finally held for 15 min. Fatty acids were compared by their retention times relative to authentic standards (FAME37 Restek 35077) and mass spectra confirmed with the National Institute of Standards and Technology database (45). The presence and synthesis of 18:3n-6 and 18:4n-6 were examined by comparison of the fragmentation spectra within the predicted retention time window of authentic fatty acid standards.

In this study, [d₅] or [1-¹³C] enrichment of n-6 or n-3 PUFA, respectively, was measured by GC-thermal conversion or combustion–isotope ratio mass spectrometry as described (46). Briefly, FAMEs were reconstituted in 25 µl of hexane and a 2-µl injection volume in splitless mode onto a Supelcowax 10 capillary column (30 m length × 0.25 mm diameter × 0.25 μm film thickness; Supelco) run with a column flow of 1.5 ml/min on a Thermo Trace 1310 gas chromatograph (ThermoFisher, Loughborough, UK) equipped with a high-temperature (1,000°C/1,400°C) combustion/thermal conversion furnace and a Thermo Delta V IRMS. The ¹³C/¹²C and ²H/¹H ratios for identified fatty acids were measured relative to laboratory reference gas standards calibrated to the international standards (Vienna Standard Mean Ocean Water or Vienna Pee Dee Belemnite, respectively). Stable isotope enrichment was calculated as described (47). The concentration of each labelled fatty acid was calculated from the amount measured by GC-FID normalised to 1 million cells.

The supernatant from unstimulated or mitogen-stimulated T cells cultured for 48 h in medium containing an EFA ratio of 8:1 was collected and immediately frozen at -80°C. Free oxylipins were isolated by solid-phase extraction (SPE) (48). Briefly, supernatants (1–2 ml) were defrosted at 4°C overnight. An antioxidant mix containing butylated hydroxytoluene and EDTA (both 0.2 mg/ml), indomethacin (100 µM), and 4-[[trans-4-[[(tricyclo[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]cyclohexyl]oxy]-benzoic acid (100 µM) in methanol/water 1:1 (v/v) (40 µl) was added to the frozen supernatant (49). The internal standard [d₅](17(S)-hydroxydocosa-4,7,10,13,15,19-hexaenoic-21,21,22,22,22-d5-acid) ([d₅]17-HDHA; 20 ng) was added, and proteins were then precipitated with 750 µl of ice-cold methanol for 30 min at -20°C. Samples were acidified with 1 M HCl (10 µl) and applied to Oasis HLB (Waters) SPE cartridges, washed with 10% (v/v) methanol in water, pure water, and hexane, and oxylipins were then each eluted with 100% ethyl acetate and methanol. Eluates were pooled, dried under nitrogen, and stored in 100 µl of methanol/water 70:30 (v/v) at -20°C before liquid chromatography–mass spectrometry (LCMS) analysis within 48 h. Oxylipins were analysed with multiple reaction monitoring (MRM) using an Acquity I-class and Xevo TQS UPLC-MS/MS system (Waters). Negative electrospray ionisation parameters were as follows: 2.4 kV capillary voltage, 40 V cone voltage, 600°C desolvation temperature, 1,000 L/h desolvation flow, 150 L/h cone flow, and 7 bar nebuliser pressure. MRM transitions are shown in Supplementary Table S1 .

Lipids were separated using a Cortecs C18 (2.1 mm × 100 mm, 1.6 µm) column (Waters) with a BEH C18 VanGuard (2.1 mm × 5 mm, 1.7 µm) pre-column (Waters) at 40°C with the autosampler temperature at 10°C and a flow rate of 0.3 ml/min. The mobile phase A was 80:20 (v/v) water/acetonitrile and mobile phase B 75:25 (v/v) acetonitrile/methanol, both containing 0.02% (v/v) formic acid. The linear gradient started with 20% mobile phase B for 1 min, increased to 35% B for 2 min, and further increased to 70% B for 7 min, with a final increase to 95% B for 2 min and a hold time of 2 min until decreasing back to 20% B for 2 min with an additional 2-min conditioning phase.

The limits of detection were 9.9 pg/µl of [d₅]17-HDHA and 9.0 pg/µl of [d₄]9-hydroxy-10(E),12(Z)-octadecadienoic acid (9(S)HODE) ( Supplementary Figure S1 ). SPE recovery and quality control coefficient of variation of [d₄]9(S)-HODE were 67% ± 10% and ± 18.1%, respectively ( Supplementary Figure S1 ). Data were processed using MassHunter 4.0 (Waters). Oxylipin concentrations were calculated relative to the internal standard [d₅]17-HDHA, then normalised to supernatant volume and background-corrected with data from the 48-h cell supernatant absent of T cells, and then normalised to the number of T cells in the cell culture.

mRNA expression of genes that encode enzymes involved in the PUFA synthesis pathway was carried out as described. Briefly, total T-cell RNA was extracted using the Qiagen RNeasy Mini kit (Qiagen) combined with on-column DNase digestion (Qiagen) as instructed by the manufacturer. RNA was eluted in RNase-free water (30 μl). RNA concentration was measured, and purity was assessed using a NanoDrop1000 spectrophotometer. RNA integrity was confirmed by agarose gel electrophoresis. cDNA was synthesised by reverse transcription, and real-time RTPCR was carried out using primers listed in Supplementary Table S2 . Amplified transcripts were quantified using the standard curve method (50) and normalised to the geometric mean of the reference genes 60S ribosomal protein L13-A (RPL13A) and succinate dehydrogenase complex, subunit A, flavoprotein variant (SDHA), which were shown to be stable across culture conditions by the GeNorm method (51). ELOVL2 and ELOVL4 mRNA expression were assessed by agarose gel electrophoresis. Briefly, the respective transcripts were amplified by 40 PCR cycles. RNA from HepG2 cells or Jurkat cells was used as a reference for ELOVL2 and ELOVL4, respectively. PCR products were resolved on 2% (w/v) agarose gel containing GelRed and visualised under UV light.

Data were analysed by one-way or two-way ANOVA with single-factor effects of time after stimulation and EFA ratio and with two-factor interaction effects using SPSS version 27 (IBM SPSS Statistics for Windows, Version 27.0; IBM Corp., Armonk, NY, USA). In some experiments, cell activation was included as an additional fixed factor. Post-hoc pairwise comparisons were done by Tukey’s test. Statistical significance was assumed at p < 0.05. The magnitude of the effect size (ηp²) was ≥0.14 for all statistically significant single-factor and interaction effects. Comparisons between unstimulated and stimulated cultures within each time point and EFA ratio were by Student’s paired t-test adjusted for multiple comparisons by the Holm–Sidak method using GraphPad Prism (Version 8 for Windows, GraphPad Software, San Diego, CA, USA; www.graphpad.com).

There was a significant single-factor effect of time [F(2,53) = 10.44, p = 0.04] and stimulation [F(2,53) = 63.23, p < 0.0001], but no significant time * stimulation interaction (p = 0.63), on CD69 expression ( Figure 1A ). The CD69 index (the ratio of the mean fluorescence intensity to the number of events in the positive region) was significantly greater in stimulated than unstimulated cells at all time points measured and increased significantly between 14 and 24 h.

FADS1 mRNA expression increased between 14 and 24 h and was significantly greater in stimulated than unstimulated cells at 24 and 48 h ( Figure 1B ). There were significant single-factor effects of time [F(2,54) = 2.17, p = 0.01] and cell stimulation [F(2,54) = 13.69, p = 0.001] and a significant time * stimulation interaction [F(2,54) = 1.2, p = 0.03] on FADS1 mRNA expression. FADS2 expression was significantly greater in stimulated than unstimulated cells at 24 and 48 h and increased significantly between 14 and 24 h. There were significant single-factor effects of time [F(2,54) = 12.41, p < 0.0001] and cell stimulation [F(2.54) = 50.38, p < 0.0001] and a significant time * cell stimulation interaction effect [F(2,54) = 11.8, p < 0.0001] on FADS2 mRNA expression ( Figure 1C ). There were no significant single-factor effects of time or cell stimulation on ELOVL5 mRNA expression ( Figure 1D ). ELOVL2 expression was below the level of detection by real-time RTPCR and agarose gel electrophoresis ( Figure 2 ). ELOVL4 expression was also below the level of detection by real-time RTPCR, although a faint band of molecular weight that corresponded to the PCR product of ELOVL4 was detected by agarose gel electrophoresis ( Figure 2 ).

Stimulation with Con A increased the amounts of the monounsaturated fatty acids 16:1n-7, 18:1n-9, and 18:1n-7 compared to unstimulated cells cultured in the presence of EFA ratios of 5:1 or 8:1 but did not alter the amounts of saturated fatty acids or 20:1n-9 ( Table 2 ). The amount of 18:2n-6 was significantly greater (approximately 2-fold) in stimulated cells than unstimulated cells at 48 h regardless of the EFA content of the medium. The amounts of 20:3n-6 and 20:4n-6 were significantly greater after 48 h (approximately 1.2-fold and 1.1-fold, respectively) regardless of the EFA ratio in the culture medium ( Table 2 ). There was no significant effect of mitogen stimulation on the amount of 20:2n-6 or 22:4n-6.

The amount of 18:3n-3 in stimulated cells was significantly (2.6-fold) greater after 48 h in medium containing an EFA ratio of 5:1 and 1.5-fold greater in medium containing an EFA ratio of 8:1 compared to unstimulated cells ( Table 2 ). The amount of 20:5n-3 was significantly greater (1.5-fold) in stimulated than unstimulated cells after 48 h, but not at 14 or 24 h, in medium containing an EFA ratio of 5:1 but did not differ significantly between mitogen-stimulated and unstimulated cells maintained in medium with an EFA ratio of 8:1 ( Table 2 ). There was no significant effect of mitogen stimulation on the amounts of 20:3n-3, 20:4n-3, 22:5n-6, or 22:6n-3 ( Table 2 ). 18:4n-3 and 18:3n-6 were not detected by either GCFID or GCMS ( Figure 3 ).

There were significant effects of duration of incubation and the EFA ratio on the change in the amounts of individual PUFA in stimulated cells compared to unstimulated cells. For n-6 PUFA, the activation-induced change in the amount of 18:2n-6 increased by approximately 65-fold with greater incubation time [F(2,54) = 12.58, p < 0.0001], but there was no significant single-factor effect of the EFA ratio (p = 0.99) or time * EFA ratio interaction (p = 0.54) ( Figure 4A ). There were no significant single-factor effects of time (p = 0.22) or EFA ratio (p = 0.11) on the activation-induced change in the amount of 20:2n-6 (time, p = 0.43; EFA ratio, p = 0.23), 20:3n-6 (time, p = 0.72; EFA ratio, p = 0.54), 20:4n-6 (time, p = 0.09; EFA ratio, p = 0.99), or 22:4n-6 (time, p = 0.47; EFA ratio, p = 0.83).

For n-3 PUFA, there was a significant single-factor effect of time [F(2,54) = 5.32, p = 0.008), such that the activation-induced change in the amount of 18:3n-3 increased by approximately 8-fold between 14 and 24 h incubation, but there was no significant effect of EFA ratio (p = 0.74) or time * EFA ratio interaction (p = 0.34) ( Figure 4B ). The activation-induced change in the amount of 20:5n-3 increased by approximately 2-fold between 14 and 48 h incubation [time F(2,54) = 5.81, p = 0.005], but there was no significant effect of the EFA ratio (p = 0.32) or time * EFA ratio (p = 0.16) interaction. There were no significant single-factor effects of time or EFA ratio on the activation-induced change in the amount of 20:3n-3 (p = 0.18 and 0.21, respectively), 20:4n-3 (p = 0.92 and p = 0.08, respectively), 22:5n-3 (p = 0.09 and p = 0.24, respectively), and 22:6n-3 (p = 0.11 and p = 0.15, respectively).

There was no significant difference in the uptake of [d₅]18:2n-6 between unesterified and ethyl-18:2n-6 in unstimulated (p = 0.9998) or stimulated T cells (p = 0.9993) ( Supplementary Figure S2 ). There was no significant difference in the conversion of [d5]18:2n-6 to [d₅]20:2n-6 between unstimulated (p = 0.1424) and stimulated T cells (p = 0.9993) ( Supplementary Figure S2 ).

The amount of labelled PUFA in stimulated T cells was approximately 1/1,000th of the total amount of each PUFA ( Tables 2 , 3 ). For the n-6 series, [d₅]18:2n-6, [d₅]20:2n-6, [d₅]20:3n-6, and [d₅]20:4n-6 were detected in unstimulated and stimulated T cells ( Table 3 and Figure 5 ). Here, [d₅]18:2n-6 was the predominant labelled n-6 fatty acid at all time points measured regardless of the EFA ratio and cell activation ( Table 2 ). Mitogen stimulation significantly increased the amount of [d₅]18:2n-6 at 24 and 48 h by approximately 2-fold each regardless of the EFA ratio in the culture medium ( Table 3 ). Mitogen stimulation increased the amount of the [d₅]18:2n-6 elongation product [d₅]20:2n-6 by 4.5-fold at 24 h and at 48 h regardless of the EFA ratio ( Table 3 ). There was no significant effect of mitogen stimulation on the amount of [d₅]20:3n-6 or [d₅]20:4n-6 in cells cultured in medium containing an EFA ratio of 5:1 ( Table 3 ). However, mitogen stimulation increased the amount of [d₅]20:3n-6 (1.6-fold) and [d₅]20:4n-6 (1.2-fold) in cells cultured in medium containing an EFA ratio of 8:1 for 48 h ( Figure 5 and Table 4 ).

There was a significant effect of time but no single-factor effect of the EFA ratio nor time * EFA ratio interaction, such that the activation-induced change in the amount of [d₅]18:2n-6, [d₅]20:3n-6, and [d₅]20:4n-6 increased by 63-fold, 7.5-fold, and 4.1-fold between 14 and 48 h, respectively ( Table 3 ). There were significant single-factor effects of time and EFA ratio and a significant time * EFA ratio interaction on the activation-induced change in the amount of [d₅]20:2n-6 ( Table 3 ). The activation-induced change in the amount of [d₅]20:2n-6 increased approximately 65-fold between 14 and 24 h in cells maintained in medium containing an EFA ratio of 5:1 and 185-fold in cells cultured in medium with an EFA ratio of 8:1 ( Figure 5 and Table 4 ).

[¹³C]18:3n-3 was the predominant labelled n-3 fatty acid at all time points measured regardless of the EFA ratio and activation state ( Table 3 ). Here, [¹³C]18:3n-3, [¹³C]20:3n-3, and [¹³C]20:5n-3 were detected in T cells regardless of the activation state ( Table 3 ). Mitogen stimulation significantly increased the amount of [¹³C]18:3n-3 at 24 and 48 h, but not at 14 h, regardless of the EFA ratio in the culture medium ( Table 3 and Figure 6 ). There was no significant effect of mitogen stimulation on the amount of [¹³C]20:3n-3 at any time point measured regardless of the EFA ratio in the culture medium ( Table 3 ). However, mitogen stimulation increased the amount of [¹³C]20:5n-3 above that of unstimulated cells cultured in medium with an EFA ratio of 5:1 at 48 h, but there was no significant effect of mitogen stimulation on the amount of [¹³C]20:5n-3 in cells cultured in medium containing an EFA ratio of 8:1 ( Figure 6 and Table 4 ).

There were significant single-factor effects of time and EFA ratio on activation-induced change in the amount of [¹³C]20:3n-3 and of time on the activation-induced change in the amount of [¹³C]20:5n-3 ( Figure 6 and Table 3 ). [¹³C]18:4n-3, [¹³C]20:4n-3, [¹³C]22:5n-3, and [¹³C]22:6n-3 were not detected after 48-h culture ( Tables 3 , 4 ).

[¹³C]9-Hydroxyoctadecatrienoic acid ([¹³C]9-HOTrE) was the most abundant of the labelled oxylipins that were measured in the T-cell culture supernatant containing an EFA ratio of 5:1, followed by lower concentrations of 9-HODE, 13-HODE, 13-HOTrE, 9,10-DiHOME, 12,13-DiHOME, 9,10-DiHODE, 12,13-DiHODE, and 15,16-DiHODE ( Figures 7A, B and Supplementary Figure S3 ). There were no significant single-factor or interaction effects of cell stimulation and EFA ratio on the concentrations of either [d₅]9-HODE (stimulation, p = 0.461; EFA ratio, p = 0.129) or [d₅]13-HODE (stimulation, p = 0.177; EFA ratio, p = 0.205) in the T-cell supernatant ( Figure 7A ). However, [d₅]9,10-DiHOME concentration was 3.2-fold greater [F(1,18) = 13.65, p = 0.002] in the supernatant from stimulated cultures with an EFA ratio of 5:1 compared to 8:1 ( Supplementary Figure S3A ). There were no significant single-factor or interaction effects of cell activation on [d₅]9,10-DiHOME. Here, [d₅]12,13-DiHOME concentration in the culture supernatant with an EFA ratio of 5:1 from stimulated cells was 1.6-fold [F(1,18) = 5.53, p = 0.03] greater than from unstimulated cultures. In addition, [d₅]12,13-DiHOME concentration in supernatants with an EFA ratio of 5:1 from mitogen-stimulated cells was 3.3-fold greater [F(1,18) = 14.66, p = 0.001] than supernatants with an EFA ratio of 8:1 ( Supplementary Figure S2A ).

There were no significant single-factor effects of EFA ratio on [¹³C]9-HOTrE, [¹³C]13-HOTrE, or DiHODE concentrations in the culture supernatants ( Figure 7B and Supplementary Figure S2B ). Here, [¹³C]9-HOTrE concentration was 6.8-fold greater [F(1,18) = 79.99, p < 0.001] in supernatants from cultures with an EFA ratio of 5:1 than 8:1. A similar effect was seen in the [¹³C]13-HOTrE concentration with a 7.5-fold increase [F(1,18) = 71.18, p < 0.001] in supernatants from cultures with an EFA ratio of 5:1 compared to 8:1. DiHODE concentrations were significantly greater in cultures with an EFA ratio of 5:1 than 8:1 [F(1,18) = 78.66, p < 0.001; Supplementary Figure S3B ].

The tracer-to-tracee ratio (TTR) was calculated for [d₅]18:2n-6 and its metabolites, namely, [d₅]20:2n-6, [d₅]9-HODE, and [d₅]13-HODE, to investigate the relative contribution of recently internalised EFA compared to the preexisting EFA pools in T cells to the synthesis of LCPUFA and oxylipins ( Figure 7C ). Freshly prepared cell media with an EFA ratio of 8:1 and containing [d₅]18:2n-6 tracer that had been stored at 4°C had a TTR of 0.0255. In contrast, intracellular [d₅]18:2n-6 from activated T cells collected after 48-h incubation had a TTR of 0.0012% ± 4%. This is equivalent to a 20-fold dilution of [d₅]18:2n-6 by endogenous unlabelled 18:2n-6. Intracellular [d₅]20:2n-6 had a 1.65-fold (p = 0.049; Sidak’s paired multiple comparison) higher TTR (0.00198 ± 14%) compared to [d₅]18:2n-6 after 48-h stimulated cell culture.

Culture media with an EFA ratio of 8:1 and containing the [d₅]18:2n-6 tracer that had been incubated at 37°C in the absence of cells had detectable amounts of (non-enzymatically) oxidised [d₅]9-HODE and [d₅]13-HODE with TTR values of 0.0372 ± 13% and 0.0391 ± 9%, respectively. The TTR of [d₅]9-HODE and [d₅]13-HODE in the supernatant from T-cell cultures incubated for 48 h were 0.0347 ± 8% and 0.0358 ± 7%, respectively, which was similar to the TTR of HODEs in 48-h cultured media without cells (p > 0.05; Sidak’s unpaired multiple comparisons) but higher than recently internalised 18:2n-6 (p < 0.001) ( Figure 7C ). Non-enzymatically formed 9-HODE in the control culture media (n = 3, 12 nmol/L) accounted for approximately one-third of total 9-HODE quantified in the 48-h T-cell supernatant and represents therefore a significant difference in concentrations [n = 10, 39 nmol/L; one-way ANOVA F(4,28) = 24.4, p = 0.002] ( Supplementary Figure S4 ).

The amount of [d₅]HODEs and its dihydroxy metabolite, namely, [d₅]DiHOME, was normalised to the intracellular [d₅]18:2n-6 substrate pool ( Table 3 ) and displayed as log2 fold change (oxylipin/18:2n-6). There was a significant effect of stimulation on the change in [d₅]9-HODE [F(1,18) = 56.13, p < 0.001] and [d₅]13-HODE [F(1,18) = 34.82, p < 0.001] concentrations relative to the 18:2n-6 pool in the supernatants from 48-h T-cell cultures for both EFA ratios. There was no significant effect of the EFA ratio on the change in the concentrations of [d₅]9-HODE [F(1,18) = 0.34, p = 0.569] and [d₅]13-HODE [F(1,18) = 0.10, p = 0.754] in supernatants from unstimulated and stimulated cells ( Figure 7D ). There was no significant effect of stimulation on the change in [d₅]9,10-DiHOME (p = 0.1) and [d₅]12,13-DiHOME (p = 0.41) concentrations, while there was a significant effect of EFA ratio on the change in [d₅]9,10-DiHOME [F(1,18) = 34.25, p < 0.001] and [d₅]12,13-DiHOME [F(1,18) = 19.77, p < 0.001] ( Figure 7D ) concentrations.