Determination of acrolein generation pathways from linoleic acid and linolenic acid: increment by photo irradiation

Acrolein is a highly reactive toxic aldehyde implicated in adverse health outcomes, including neurodegenerative and cardiovascular diseases. Thermal degradation of edible oils is a common exposure source. Earlier work posited triacylglycerol glycerol backbones as precursors, but isotope studies indicated this is not predominant; instead, oxidation of fatty acids, particularly α-linolenic acid (LnA), was implicated under heat, with much lower contributions from linoleic acid (LA) and negligible from oleic acid (OA). Prior studies, however, largely examined radical oxidation pathways that generate specific fatty acid hydroperoxide (FAOOH) positional isomers. Recent evidence shows that singlet oxygen (1O2)-mediated photo-oxidation contributes significantly to oxidation in commercial oils, producing distinct FAOOH positional isomers from those formed by radical pathways. Given that acrolein formation depends on FA species and hydroperoxyl group position, the study aims to reassess acrolein generation pathways under 1O2 photo-oxidation, testing whether 1O2-derived FAOOH of LA and LnA are important acrolein sources and whether photo-irradiation increases acrolein formed upon heating edible oils.

- Early studies proposed acrolein formation from glycerol dehydration after triacylglycerol hydrolysis, but isotope labeling indicated glycerol is not the main source. - More recent work showed fatty acid oxidation, especially from LnA, is a major acrolein source during heating; acrolein from LnA can be ~10-fold higher than from LA, with OA contributing negligibly. - Radical oxidation of FA produces specific FAOOH positional isomers (e.g., for LnA: 9-, 12-, 13-, 16-HpOTE), which undergo β-scission leading to aldehydes. - Singlet oxygen (1O2) photo-oxidation also occurs in edible oils during storage/exposure to light, generating different FAOOH positional isomers (e.g., 10- and 12-HpODE from LA; 10- and 15-HpOTE from LnA) compared with radical oxidation. - Prior analyses of commercial oils detected significant amounts of 1O2-specific FAOOH immediately after opening, highlighting the need to evaluate acrolein formation from photo-oxidation pathways.

- Preparation of FAOOH isomer standards: - OA oxidized via radical initiation using azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) at 45 °C for 6 h; crude products purified by reverse-phase HPLC to isolate HpOME, followed by normal-phase HPLC to separate 8-, 9-, 10-, and 11-HpOME isomers. - LA and LnA oxidized via type II photo-oxidation using rose bengal (500 µg in 50 mL methanol) under LED irradiation (50,000 lux, 4 °C, 15 h) to form HpODE and HpOTE; rose bengal removed with QMA SPE; isomers purified by normal-phase semipreparative HPLC. - Purity and identity checked by TOF-MS: sodiated adduct [M+Na]+ exact masses matched theory (HpOME m/z 337.2355; HpODE m/z 335.2198; HpOTE m/z 333.2042). Hydroperoxyl positions confirmed via product ion scans producing position-specific fragments. Concentrations determined by FOX assay. - Thermal decomposition of FAOOH isomers: - 50 nmol of each FAOOH dried in 2 mL amber vials, sealed in air; heated at 180 °C for 30 s (n=3), then cooled on ice. Complete decomposition confirmed. - Volatile products sampled from headspace using SPME (DVB/CAR/PDMS fiber, 20 min at 40 °C) and analyzed by GC-EI-MS (DB-WAX-UI column). Temperature program for standards: 30 °C (10 min), ramp 5 °C/min to 250 °C, hold 5 min. Compounds identified via NIST 17 library and standards (acrolein). - Photo-irradiation and heating of marketed edible oils: - Rapeseed, rice bran, and soybean oils (1 g) placed in transparent vials (n=1 per condition) and irradiated under LED (5000 lux, 16–18 °C) for 0–7 days to simulate shelf storage; then heated at 180 °C for 90 s; volatiles analyzed by SPME GC-EI-MS with a faster GC program for oils (30 °C 10 min, ramp 50 °C/min to 250 °C, hold 15 min). - Data analysis: - Volatiles cataloged (aldehydes, alcohols, ketones, furans, hydrocarbons). Acrolein quantified by GC peak area; replicate data reported as mean ± SD (n=3) for standards.

- OA hydroperoxides (HpOME isomers) did not generate detectable acrolein upon thermal decomposition, consistent with minimal acrolein from trioleate. - LA hydroperoxides: - Radical-type HpODE isomers (9- and 13-HpODE) did not yield acrolein. - 1O2-specific HpODE isomers (10- and 12-HpODE) generated significant acrolein, newly identifying LA as a source of acrolein under photo-oxidation. - LnA hydroperoxides (HpOTE): - All HpOTE isomers produced acrolein on decomposition. - 1O2-specific HpOTE isomers (10- and 15-HpOTE) produced approximately 2–3 times more acrolein than radical-type isomers (9-, 12-, 13-, 16-HpOTE). The study notes about twofold higher amounts for 10-/15-HpOTE vs others. - Mechanistic support: - Proposed pathways involve initial O–OH bond homolysis to alkoxyl radicals, β-scission favoring formation of conjugated/stabilized products, radical delocalization, and peroxidation to 3-hydroperoxy-1-alkenes that undergo further β-scission to release acrolein. - Detected byproducts consistent with pathways, including 2-octenal, 2-octen-1-ol, 1-octen-3-one, 1-octen-3-ol, 1-pentanol, pentanal (from 10-HpODE) and 2-heptenal, octanoic acid (from 12-HpODE). - Photo-irradiated edible oils: - Oils stored in the dark did not produce acrolein upon heating. - Photo-irradiation (5000 lux) increased acrolein generated upon subsequent heating, with increases evident in oils higher in LA content (e.g., rice bran, soybean), supporting the role of 1O2-specific HpODE/HpOTE in acrolein formation.

The study demonstrates that acrolein formation depends strongly on the fatty acid class and the hydroperoxyl group position of FAOOH. Contrary to assumptions based solely on radical oxidation, LA becomes a significant acrolein precursor when oxidized by singlet oxygen, via 10- and 12-HpODE. For LnA, while all HpOTE isomers yield acrolein, the 1O2-derived 10- and 15-HpOTE generate substantially more acrolein, consistent with mechanistic predictions that these isomers form the key -CH=CH-CH(OOH)-CH2-CH=CH- motif twice during decomposition, producing two acrolein molecules. The detection of conjugated aldehydes and related alcohols/ketones supports β-scission, radical delocalization, and 3-hydroperoxy-1-alkene intermediates as critical steps. Together with increased acrolein after photo-irradiation of edible oils, these findings establish a photo-oxidation-driven pathway for acrolein generation and highlight light exposure as a practical driver of acrolein risk in oils, complementing known thermal radical pathways.

This work identifies a previously underappreciated pathway for acrolein formation in edible oils: decomposition of singlet oxygen (1O2)-specific FA hydroperoxides from LA (10-, 12-HpODE) and LnA (10-, 15-HpOTE). OA-derived hydroperoxides do not contribute. 1O2-specific HpOTE produce 2–3-fold more acrolein than radical-derived HpOTE, and photo-irradiation of oils increases acrolein formed upon heating, especially in LA-rich oils. These insights inform strategies to minimize acrolein exposure by limiting light exposure during oil storage and handling. Future research should structurally characterize unidentified volatile products, quantify acrolein yields under diverse culinary conditions, extend analyses to other PUFA-rich oils and matrices, and evaluate packaging/antioxidant interventions to mitigate photo-oxidation.

- The mechanistic pathway presented represents one of several possible decomposition routes (e.g., alternative peroxide rearrangements such as Hock fragmentation may occur). - Several intense GC-MS peaks from HpOTE decompositions remained unidentified, limiting complete mechanistic validation. - Marketed oil irradiation experiments used n=1 per condition, reducing statistical power and generalizability. - Thermal decomposition was conducted in sealed small vials at 180 °C for short durations, which may not fully replicate all real-world cooking scenarios. - The study focuses on specific purified FAOOH isomers; in complex food systems, interactions with other constituents (antioxidants, metals) may alter pathways and yields.