Ole, oleic acid (18:1, cis-9-octadecenoic acid); Lin, linoleic acid (18:2, cis,cis-9,12-octadecenoic acid); 11,11-D₂-Lin (11,11-D₂-18:2; 11,11-D₂-cis,cis-9,12-octadecenoic acid); 8,8-D₂-Lin (8,8-D₂-18:2; 8,8-D₂-cis,cis-9,12-octadecenoic acid); 11,11-D,H-Lin (11,11-D,H-18:2; 11,11-D,H-cis,cis-9,12-octadecenoic acid); 11-¹³C-Lin (11-¹³C-18:2; 11-¹³C-cis,cis-9,12-octadecenoic acid); αLnn, linolenic acid (18:3, cis,cis,cis-9,12,15-octadecenoic acid); 11,11,14,14-D₄-αLnn (D₄-18:3; 11,11,14,14-D₄-cis,cis,cis-9,12,15-octadecenoic acid).

Ratios of trans,cis-/trans,trans- HODEs versus concentration of ethyl linoleate (D₀ or D₂) were plotted. Oxidations were carried out at 37 °C in benzene for 1 h and initiated with MeOAMVN. HODE ethyl esters were analyzed by HPLC-UV (250 × 4.6 mm silica column; 5 µm; elution solvent: 0.5% 2-propanol in hexanes; monitoring wavelength: 234 nm).

(A) Typical chromatogram of HPLC-UV (250 × 4.6 mm silica column; 5 µm; elution solvent: 1.4% isopropanol, 0.1% acetic acid in hexanes; 1.0 mL/min; monitoring wavelength: 234 nm; analysis of D₀-Lin: D₂-Lin co-oxidation products. (B) Typical chromatogram from HPLC-MS analysis (150 × 4.6 mm silica column; 3 µm; elution solvent: 1.4% isopropanol, 0.1% acetic acid in hexanes; 1.0 mL/min) of D₀-Lin: D₂-Lin co-oxidation. The first two panels are monitoring for D₀-fragmentation ions. The last two panels are monitoring for D₁-fragmentation ions (which have a m/z value corresponding to a mono-deutero ion). (C) Total HODEs vs. percentage of 11,11-D₂-Lin in co-oxidation experiments. Co-oxidation mixtures of D₀-Lin and D₂-Lin were analyzed by HPLC-UV as shown in panel A. Total amount of HODEs were quantified relative to the level of the internal standard, 4-methoxybenzyl alcohol, present in the sample.

(A) Wild-type, yeast Q-less coq3, or respiratory deficient cor1 null mutants were harvested during mid-log phase growth (0.2–1.0 OD₆₀₀). Yeast cells were washed twice with sterile water and diluted to 0.2 OD₆₀₀ in phosphate buffer. Yeast cells were treated with 200 µM of the designated fatty acid. Serial dilutions (1:5) starting at 0.2 OD/ml were spotted on YPD solid plate medium. A zero-time untreated control is shown on the top left. Pictures were taken after 2 days of growth at 30 °C. (B) Yeast coq3 null mutant cells were treated with the designated fatty acid for 2 hours and three 100 µl aliquots were removed and spread onto YPD plate medium after dilution. The chart shows the number of colony forming units (CFU) per µl after 2 hours of PUFA treatment. There was no significant difference between CFU of different treatments. (C) After 2 hours of PUFA treatment coq3 null mutant cells were treated with 8 µM C11-BODIPY 581/591 for 30 min at room temperature. Four 100 µl aliquots were plated in a 96-well plate and the fluorescence was measured by fluorimetry with a 485 nm excitation and a 520 nm emission filter using a Perkin Elmer, 1420 Multilabel counter, Victor3 in 5 min increments following 30 min of C11-BODIPY 581/591 treatment. Fatty acid treatments include: no treatment, blue; Lin, black; 8,8-D₂-Lin, green; 11,11-D₂-Lin, purple. (D) Lipid peroxidation in cells treated with the designated fatty acid was examined as described in (C) except cells were visualized by fluorescent microscopy using excitation at 490 nm with a 520 nm emission filter within 45 min after C11BODIPY 581/591 treatment. Lipid peroxidation was visualized by an Olympus IX70 fluorescence microscope with 100X oil objective and a 490 nm excitation and a 520 nm emission filter (FITC). (Scale bar= 6.6 µm).

(A) Fatty acid sensitivity assays were performed as described in , except yeast cells were treated with 200 µM of the designated fatty acid for 10 h. A zero-time untreated control is shown in the top left. Pictures were taken after 2 days of growth at 30 °C. (B) Coq3 null mutant cells were treated with the designated fatty acid for 3 hours and three 100 µl aliquots were removed and spread onto YPD plates after dilution. The chart shows the number of colony forming units (CFU) per µl after 3 hours of PUFA treatment. (C) After 3 hours of PUFA treatment coq3 null mutant cells were treated with 8 µM C11-Bodipy 581/591 for 30 min at room temperature. Four 100 µl aliquots were plated in a 96-well plate and the fluorescence was measured as described in . Fatty acid treatments include: Ole, green; Lin, black; 11,11-D,H-Lin, purple; 11,11-D₂-Lin, blue.

(A) Fatty acid sensitivity assays were performed as described in except that yeast were treated with 200 µM of the designated PUFA mixture (natural PUFA : isotope-reinforced PUFA) for either 3.5 or 10 hours. A zero-time untreated control is shown on the top left. Pictures were taken after 2 days of growth at 30 °C. (B) Yeast coq3 null mutant cells were treated with the designated fatty acid mixture for 30 min and three 100 µl aliquots were removed and spread onto YPD plates after dilution. The chart shows the number of colony forming units (CFU) per µl after 30 min of PUFA treatment. There was no significant difference between CFU of different treatments. (C) Yeast coq3 null mutant cells were treated with 8 µM C11-Bodipy 581/591 for 30 min at room temperature. Four 100 µl aliquots were plated in a 96-well plate and the fluorescence was measured as described in . Fatty acid treatments include: 100% αLnn, black; 20% D₄-αLnn, purple; 100% D₄-αLnn, light blue.

Fatty acid sensitivity assays were performed as described in except that yeast were treated with 200 µM of the designated PUFA mixture (natural PUFA : isotope-reinforced PUFA) for either 4 or 10 hours. A zero-time untreated control is shown on the top left. Pictures were after taken 2 days of growth at 30 °C.

(A) Fatty acid sensitivity assays were performed as described in except that yeast were treated with 200 µM Ole or 100 µM of αLnn in the presence of 100 µM of the designated fatty acid for 4 hours. A zero-time untreated control is shown on the top left. Pictures were taken after 2 days of growth at 30 °C. (B) Fatty acid sensitivity assays were performed as described in except that yeast were treated with 300 µM Ole or 300 µM of Lin in the presence of 300 µM of the designated fatty acid for 10 hours. A zero-time untreated control is shown on the top left. Pictures were taken after 2 days of growth at 30 °C.

(A) Wild-type yeast cells were treated as described in except yeast were treated with 200 µM of the designated fatty acid for 2 hours, washed with sterile water, and were either not treated (triangles) or treated with 50 µM CuSO₄ (squares) at room temperature. After 60 min of copper treatment cells were treated with 8 µM C11-Bodipy 581/591 for 30 min at room temperature. Four 100 µl aliquots were plated in a 96-well plate and the fluorescence was measured as described in . Fatty acid treatments include: no treatment, blue; Lin, black; 8,8-D₂-Lin, green; or 11,11-D₂-Lin, purple. (B) The chart shows the fluorescence intensity per OD₅₉₅ at the 60 min time point shown in (A) and corresponds to no copper (white bars) or copper treatment (black bars). Wild-type yeast cells treated with copper in the absence or presence of PUFA have significantly higher levels of lipid peroxidation as compared to yeast not treated with copper; a, p<1.0E–4. Wild-type yeast cells treated with copper in the presence of Lin or 8,8-D₂-Lin have significantly higher levels of lipid peroxidation as compared to yeast treated with copper in the absence of PUFA; b, p<1.0E–4. Differences in fluorescence signals were assessed with one-way ANOVA test followed by a Tukey comparison test (Stat View 5.0.1, SAS, CA). (C) Lipid peroxidation of the designated fatty acid was examined as described in except cells were visualized by fluorescent microscopy after 60 min of 50 µM CuSO₄ treatment. (Scale bar = 5 µm).

Wild-type cells were treated as described in except yeast were treated with the designated fatty acid for 3 hours, washed with sterile water, and treated with 8 µM C11-BODIPY 581/591 for 30 min at room temperature. Following BODIPY 581/591 treatment wild-type yeast were either not treated (white bars) or treated with 50 µM CuSO₄ (black bars) at room temperature. The chart shows the fluorescence intensity per OD₅₉₅ after 60 min of either no copper (white bars) or plus copper (black bars) treatment. Wild-type yeast treated with copper in the absence or presence of PUFAs have significantly higher levels of lipid peroxidation as compared to yeast not treated with copper; a, p<1.0E–4. Wild-type yeast treated with copper in the presence of Lin have significantly higher levels of lipid peroxidation as compared to yeast treated with copper in the presence of Ole; b, p < 1.0E–4. Differences in fluorescence signals were assessed with one-way ANOVA test followed by a Tukey comparison test (Stat View 5.0.1, SAS, CA).

Fatty acid sensitivity assays were performed as described in except that yeast were treated with 300 µM of the designated PUFA or PUFA mixture for 10 hours. A zero-time untreated control is shown on the top left. Picture were taken after 3 days of growth at 30 °C.

A theoretical chain reaction is depicted where a single initiation event producing a lipid peroxyl radical (denoted by –OO•) starts a chain reaction of lipid autoxidation that in the presence of O₂, may continue indefinitely (A) (red arrows), and produce many molecules of lipid peroxides; susceptible phospholipid molecules containing a PUFA acyl chain are designated by a kinked blue line and a red dot. (B) Propagation of PUFA autoxidation can progress by interaction with any neighboring PUFAs. (C) The presence of 20% isotope-reinforced PUFA (denoted by a black kinked line and a black dot) inhibit (or slow) chain propagation. (D) Propagation is inhibited for PUFAs neighboring the D-PUFA.