Supplies
DHA and diolein (DG 18:1_18:1) had been bought from Nu-Examine Prep, Inc (Elysian, MN, USA). Rose bengal, butyl hydroxytoluene (BHT), N,N’-dicyclohexylcarbodiimide (DCC), dimethylaminopyridine (DMAP), and 2-methoxypropene (MxP) had been obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Pyridinium p-toluenesulfonate (PPTS) was obtained from Sigma-Aldrich (St. Louis, MO, USA). 1-Palmitoyl-lysophosphatidylcholine (LPC 16:0/0:0) was obtained from Avanti Polar Lipids (Alabaster, AL, USA). All different reagents had been of the very best grade out there. Recent uncooked mackerel fillet was bought from a neighborhood grocery retailer in Sendai, Japan.
Extraction of impartial and polar lipids in mackerel
Whole lipid was extracted from uncooked mackerel (300 mg) utilizing the modified Folch extraction technique17,18. The obtained complete lipid fraction was dissolved in 600 µL of chloroform/2-propanol (2:1, v/v). Whole lipid (300 µL) was loaded onto an aminopropyl cartridge (Strata NH2, 55 µm, 70 Å, 100 mg, Phenomenex Inc. CA, USA), which was equilibrated with chloroform/2-propanol (2:1, v/v), and additional eluted with chloroform/2-propanol (2:1, v/v, 2 mL). The eluent was collected as impartial lipids. Then, methanol (2 mL) was utilized to elute phospholipids. The collected impartial lipids and phospholipids had been dried and dissolved in 500 µL of hexane and 500 µL of methanol, respectively. These samples had been saved at − 80 °C till the evaluation. Previous to storage, the headspace of every tube was crammed with N2 gasoline to stop additional oxidation.
Evaluation of the phospholipids extracted from mackerel
The extracted phospholipid fraction was diluted 1000-fold with methanol, and 1 µL of the answer was subjected to liquid chromatography (LC)-MS/MS consisting of ExionLC system (SCIEX, Tokyo, Japan) outfitted with a 4000 QTRAP mass spectrometer (SCIEX). Separation was carried out by isocratic elution with methanol/water (95:5, v/v) on a COSMOSIL Packed Column (5C18-MS-II, 5 μm, 2.0 ID × 250 mm, Nacalai Tesque Inc., Kyoto, Japan) at 45 °C. The stream charge was 0.2 mL/min. By mixing the column eluate with sodium acetate answer19, PC was detected as Na+ adduct in Q1 mass scan mode. Considerable PC molecular species had been chosen primarily based on a Q1 mass scan, and their constituent fatty acids had been decided by a product ion scan in direct infusion. The devices had been operated by Analyst software program (ver. 1.7.2). The MS parameters are proven in Desk S1 of Supplementary Materials.
Evaluation of the impartial lipids extracted from mackerel
The extracted impartial lipid fraction was diluted 1000-fold with methanol, and 1 µL of the answer was injected into LC–MS/MS. Separation was carried out by binary gradient elution with solvent A (methanol) and solvent B (2-propanol) on an ACQUITY UPLC HSS C18 Column (100 Å, 1.8 µm, 2.1 × 150 mm, Waters Corp, MA, USA) at 45 °C. The gradient program was as follows: 0–20 min, 20–100% B (0.2 mL/min). TG was detected as Na+ adducts in Q1 and impartial loss (NL) scan mode by mixing the column eluate with sodium acetate answer. TG molecular species in mackerel had been analyzed by setting up 2-dimensional maps (2D maps)20. TG molecular species with the identical complete carbon quantity (C) are aligned on horizontal strains whereas these with the identical complete diploma of unsaturation (n) are aligned on diagonal strains on 2D maps21. Based mostly on these analyses, the spots consisting of TG-DHA molecular species had been chosen, and the opposite constituent fatty acids had been additional decided by product ion scan. The MS parameters are proven in Desk S1 of Supplementary Materials.
Mannequin pattern preparation of a mix of PC-DHA;OOH isomers
PC 16:0/22:6 was decided as a goal because it was considerable in mackerel primarily based on the evaluation of the extracted phospholipid fraction. A mix of its hydroperoxide isomers (i.e., PC 16:0/22:6;OOH isomers) was ready as a mannequin pattern of PC-DHA;OOH.
DHA (399 mg) was esterified to LPC 16:0/0:0 (200 mg) in chloroform (26.9 mL) containing DCC (812 mg) and DMAP (234 mg) beneath N2 gasoline for 28 h at 40 °C22. After the response, the phospholipid fraction was collected with Sep-Pak Vac Silica (35 cc, 10 g, Waters, MA, USA)17. The PC 16:0/22:6 was chromatographically purified as in our earlier report23. PC 16:0/22:6 was additional purified by eradicating the salt contained within the collected fraction. The purified PC 16:0/22:6 was dissolved into methanol (20 mL).
Rose bengal (0.15 mg) was added to the PC answer, and photo-oxidation was carried out by exposing the answer to LED irradiation (6000 lx, on ice) for 3 h. Rose bengal was eliminated with Sep-Pak Accell Plus QMA (360 mg, Waters). The eluent was dried beneath N2 gasoline. PC 16:0/22:6;OOH was purified in the identical method because the PC 16:0/22:6 purification. The whole weight of obtained PC 16:0/22:6;OOH isomers was 1.6 mg after the collected eluent was dried. The pattern was dissolved in chloroform (200 μL) and saved at − 80 °C till the evaluation. The headspace of the pattern vial was crammed with N2 gasoline previous to storage.
Mannequin pattern preparation of a mix of TG-DHA;OOH isomers
TG 18:1_18:1_22:6 was decided as a goal because it was considerable in mackerel primarily based on the evaluation of the extracted impartial lipid fraction. A mix of its hydroperoxide isomers (i.e., TG 18:1_18:1_22:6;OOH isomers) was ready as a mannequin pattern of TG-DHA;OOH.
Whereas PC 16:0/22:6;OOH was ready by photo-oxidation of PC 16:0/22:6, TG 18:1_18:1_22:6 being uncovered to LED irradiation would generate TG 18:1_18:1;OOH 22:6 along with TG 18:1_18:1_22:6;OOH. Thus, the specified product (i.e., TG 18:1_18:1_22:6;OOH) was completely ready by esterification of DHA;OOH (protected with MxP) to DG 18:1_18:1.
DHA (1 g) was dissolved in 24 mL of methanol containing rose bengal (0.52 mg). The answer was uncovered to LED irradiation (6000 lx, 4 °C) for roughly 17 h. Rose bengal was eliminated by the identical process as in 2.5. The combination of DHA;OOH isomers and unoxidized DHA had been collected by a semi-preparative LC19. The publicity of unoxidized DHA to LED irradiation was repeated to additional acquire DHA;OOH. The hydroperoxyl group of the collected DHA;OOH was protected with MxP and esterified to DG 18:1_18:1 as in earlier research19,22. After deprotection, the resultant combination of TG 18:1_18:1_22:6;OOH isomers weighed 9.2 mg. The pattern was saved at − 80 °C till the evaluation. The headspace of the pattern vial was crammed with N2 gasoline previous to storage.
Picture- and thermal oxidation of mackerel
A uncooked mackerel fillet was lower into single items that weigh round 300 mg every. The fillet didn’t comprise a head or fins. The pores and skin was eliminated earlier than chopping. For photo-oxidation, a bit of the lower mackerel was put into a ten mL clear glass tube. Every tube was capped and uncovered to LED mild in a cool incubator (2000 lx, 4 °C) for twenty-four, 48, or 72 h. For thermal oxidation, a bit of the lower mackerel was put into a ten mL amber glass tube. Every tube was heated with its caps on at 100 °C on a block heater (TB-620 Sizzling Block Baths, Advantec Co., Ltd., Tokyo, Japan) for 4, 6, or 8 min. Every pattern was flipped as soon as in a tube in order that the warmth transferred evenly. Phospholipids and impartial lipids had been extracted from 50 mg of photo- and thermally oxidized mackerel in the identical method as in 2.3, and reconstructed in 500 μL of methanol and hexane, respectively. The extracted samples had been saved at − 80 °C till evaluation.
PC-DHA;OOH and TG-DHA;OOH isomers analyses within the ready mannequin samples and mackerel extract by LC–MS/MS
To develop the direct evaluation technique of PC-DHA;OOH and TG-DHA;OOH isomers, every mannequin pattern of PC 16:0/22:6;OOH isomers combination and TG 18:1_18:1_22:6;OOH isomers combination was infused into 6500 QTRAP mass spectrometer (SCIEX), to acquire Q1 mass and product ion mass spectra. To tell apart the hydroperoxide place of every isomer, their particular product ions had been chosen. Utilizing chosen product ions, a number of response monitoring (MRM) pairs had been constructed and the MS parameters had been optimized (Desk S2 and Desk S3 of Supplementary Materials). The devices had been operated by Analyst software program (ver. 1.7.2.).
Utilizing constructed MRM pairs, LC–MS/MS circumstances for PC 16:0/22:6;OOH and TG 18:1_18:1_22:6;OOH isomers had been optimized with the ExionLC system (SCIEX) outfitted with a 6500 QTRAP. Separation of PC 16:0/22:6;OOH was carried out by binary gradient elution with solvent A (water) and solvent B (methanol) on a COSMOSIL Packed Column (5C18-MS-II, 5 μm, 2.0 ID × 250 mm, Nacalai Tesque Inc.) at 40 °C. The gradient program was as follows: 0–15 min, 90–97.5% B linear at 0.2 mL/min. The column eluent was blended with a post-column solvent consisting of methanol containing 2 mM of sodium acetate at 0.01 mL/min to advertise ionization. For TG 18:1_18:1_22:6;OOH isomers, the separation was carried out by binary gradient elution consisting of solvent A (hexane/2-propanol, 100:1, v/v) and solvent B (hexane/2-propanol, 1000:5, v/v) on an Intertsil SIL-100A (3 μm, 2.1 × 250 mm, GL Sciences Inc., Tokyo, Japan) at 40 °C. The gradient program was as follows: 0–26 min, 0–86.7% A linear at 0.2 mL/min. The column eluent was blended with a post-column solvent consisting of methanol/2-propanol (4:1, v/v) containing 0.2 mM of sodium acetate at 0.1 mL/min to advertise ionization.
A portion (10 μL) of every mackerel pattern obtained in 2.7 was analyzed utilizing the optimized LC–MS/MS circumstances.