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, 53 (4), 893-906

Small Amounts of Isotope-Reinforced Polyunsaturated Fatty Acids Suppress Lipid Autoxidation

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Small Amounts of Isotope-Reinforced Polyunsaturated Fatty Acids Suppress Lipid Autoxidation

Shauna Hill et al. Free Radic Biol Med.

Abstract

Polyunsaturated fatty acids (PUFAs) undergo autoxidation and generate reactive carbonyl compounds that are toxic to cells and associated with apoptotic cell death, age-related neurodegenerative diseases, and atherosclerosis. PUFA autoxidation is initiated by the abstraction of bis-allylic hydrogen atoms. Replacement of the bis-allylic hydrogen atoms with deuterium atoms (termed site-specific isotope-reinforcement) arrests PUFA autoxidation due to the isotope effect. Kinetic competition experiments show that the kinetic isotope effect for the propagation rate constant of Lin autoxidation compared to that of 11,11-D(2)-Lin is 12.8 ± 0.6. We investigate the effects of different isotope-reinforced PUFAs and natural PUFAs on the viability of coenzyme Q-deficient Saccharomyces cerevisiae coq mutants and wild-type yeast subjected to copper stress. Cells treated with a C11-BODIPY fluorescent probe to monitor lipid oxidation products show that lipid peroxidation precedes the loss of viability due to H-PUFA toxicity. We show that replacement of just one bis-allylic hydrogen atom with deuterium is sufficient to arrest lipid autoxidation. In contrast, PUFAs reinforced with two deuterium atoms at mono-allylic sites remain susceptible to autoxidation. Surprisingly, yeast treated with a mixture of approximately 20%:80% isotope-reinforced D-PUFA:natural H-PUFA are protected from lipid autoxidation-mediated cell killing. The findings reported here show that inclusion of only a small fraction of PUFAs deuterated at the bis-allylic sites is sufficient to profoundly inhibit the chain reaction of nondeuterated PUFAs in yeast.

Conflict of interest statement

The other authors have no conflict to declare.

Figures

Figure 1
Figure 1. Structures of fatty acids used in this study
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).
Figure 2
Figure 2. Clocking experiments on Lin and 11,11-D2-Lin ethyl esters
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).
Figure 3
Figure 3. In vitro kinetic studies of PUFA autoxidation
(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.
Figure 4
Figure 4. Isotope reinforcement at the bis-allylic position of polyunsaturated fatty acids is required for protection against lipid autoxidation
(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).
Figure 5
Figure 5. Yeast coq3 null mutants treated with mono-deuterated 11,11-D,H-Lin are resistant to PUFA-induced sensitivity
(A) Fatty acid sensitivity assays were performed as described in Figure 4, 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 Fig 4C. Fatty acid treatments include: Ole, green; Lin, black; 11,11-D,H-Lin, purple; 11,11-D2-Lin, blue.
Figure 6
Figure 6. Only a small fraction of isotope-reinforced PUFAs is required for rescue
(A) Fatty acid sensitivity assays were performed as described in Figure 4 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 Figure 4C. Fatty acid treatments include: 100% αLnn, black; 20% D4-αLnn, purple; 100% D4-αLnn, light blue.
Figure 7
Figure 7. A small fraction of either D2-11,11-Lin or D4-11,11,14,14-αLnn is sufficient to rescue sensitivity of coq3 mutant yeast cells to either PUFA treatment
Fatty acid sensitivity assays were performed as described in Figure 4 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.
Figure 8
Figure 8. Yeast coq3 and coq9 null mutant cells are sensitive to a fatty acid mixture containing monounsaturated (Ole) and αLnn or Lin
(A) Fatty acid sensitivity assays were performed as described in Figure 4 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 Figure 4 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.
Figure 9
Figure 9. Isotope-reinforcement of Lin at the bis-allylic position protects copper-stressed wild-type cells from lipid peroxidation
(A) Wild-type yeast cells were treated as described in Figure 4 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 Figure 4C. 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 Figure 4D except cells were visualized by fluorescent microscopy after 60 min of 50 µM CuSO4 treatment. (Scale bar = 5 µm).
Figure 10
Figure 10. Both mono- and di-deuterated Lin at the bis-allylic position protect copper-stressed wild-type cells from lipid peroxidation
Wild-type cells were treated as described in Figure 4 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).
Figure 11
Figure 11. Small amounts of isotope-reinforced PUFAs protect yeast cells from long chain PUFA stress
Fatty acid sensitivity assays were performed as described in Figure 4 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.
Figure 12
Figure 12. Isotope-reinforced PUFAs limit the chain reaction of lipid autoxidation when present at only 20%
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.

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