In the Laboratory
Photosensitized Peroxidation of Lipids An Experiment Using 1H-NMR Marion W. Smith,* Renee Brown, Steven Smullin, and Jon Eager Muhlenberg College, Allentown, PA 18104 Naturally occurring lipids contain a mixture of unsaturated fatty acids such as oleic, linoleic, and linolenic acids. The double bonds of these species are the target for peroxidation reactions that not only lead to degradation but affect biological properties of lipids as well. For example, food scientists are concerned with the deterioration of fats in food, and cell biologists are interested in the causes of oxidation of membrane lipids, which is implicated in aging and as a possible cause for cancer. The rate of oxidation of lipids is increased in the presence of light and with certain dyes acting as photosensitizers; the oxidation is slowed or stopped in the presence of antioxidants such as vitamin E (1). Photosensitized oxidation can proceed through several pathways (2, 3). In one class of photosensitizers (represented by chlorophyll [4], erythrosin [5, 6], hematoporphyrin [6–8], zinc tetramethylpyridyl porphyrin [7], and methylene blue [6, 7]), the sensitizer transfers energy to molecular oxygen causing a transition to the singlet state. The singlet oxygen then reacts with the substrate. The Peroxidation Reaction
methyl linoleate four monohydroperoxy isomers are formed, shown in Figure 1. The isomers have been identified by separation from the reaction mixture, reduction of the hydroperoxy groups to the hydroxy derivatives, then characterization by mass spectroscopy (1, 5). HPLC separation of hydroxy derivatives has also been used to show the approximately equimolar distribution of the four isomers (6). Studies on methyl linoleate indicate that the next step involves cyclization to a 5-membered ring dioxane and attack by a second molecule of oxygen (6) as shown in Figure 2. Ultimate breakdown of lipids in biological systems includes formation of malondialdehyde (9). Methods of Determining Extent of Peroxidation Monitoring the progress of a lipid peroxidation reaction has been achieved in a number of widely used methods that give a quantitative assessment of the extent of oxidation, but give limited information on the structure of the products. Four of these methods are as follows: 1. Oxidation of acidified iodide to liberate iodine (6). 2. Formation of the thiobarbituric acid (TBA) adduct to malondialdehyde and measurement of absorbance of this adduct at 245 nm (8–10). 3. Monitoring the UV absorption at 233 nm; this increases owing to the conjugated double bonds in the product (5, 6). 4. Measuring the consumption of oxygen gas by gas buret (5, 7, 11).
Methyl linoleate (9,12-methyloctadecadieneoate) is commercially available and is a convenient model compound in studying the chemistry of unsaturated lipids. The first products from the singlet oxygen reaction with an unsaturated fatty acid ester result from “ene” addition to a carbon and shift of the double bond (2). Thus for O O
OOH R
CH3
1H
OOH R'
R
13-OOH
R' 9-OOH
NMR 1H NMR has been used to determine the structure of separated and purified lipid oxidation products of methyl oleate and linoleate (4). The chemical shift of the –OOH proton varies between 8.5 and 13 ppm depending on lipid type and concentration and organic solvent (12–14). However, other spectral features are also indicative of peroxidation. Resonances at 5.6–6.7 ppm are due to protons of the conjugated diene products (13, 14), whereas those at 4.2–4.3 ppm arise from the methine proton on the carbon with the hydroperoxy group (4, 13, 14).
1H
OOH R
OOH R'
R
12-OOH
R'
10-OOH O
R=
R' =
O
CH3
Figure 1. Methyl linoleate and hydroperoxy isomers formed from reaction with singlet oxygen.
NMR To Demonstrate Peroxidation The following experiment shows how 1H NMR can be used to monitor the reaction and thus evaluate the effectiveness of various photosensitizers in causing rapid oxidation, or to evaluate the protective effect of antioxidants. The photosensitizers that have been found effective using these methods include 5,10,15,20-tetraphenyl porphyrin (TPP), available from Aldrich, and tetra(4-hydroxy)phenyl porphyrin, available from Midcentury, Posen, IL. These can be most conveniently added to the lipid in the form of a concentrated solution in acetone. Good results can be obtained using a 60-MHz NMR spectrometer.
*Corresponding author.
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In the Laboratory OOH
O
O
OOH
Figure 2. Second step in peroxidation: cyclization of 10hydroperoxy methyl linoleate and reaction with second molecule of singlet oxygen.
The procedure used is as follows. Fifty microliters (1.5 × 10{4 mol) of methyl linoleate is placed in an NMR tube and mixed with 1 mL of deuterated acetone with TMS as internal reference, and approximately 10{8 mol of photosensitizer is added (2–3 drops of concentrated solution of TPP in deuterated acetone). This gives a pinkish purple mixture. The 1H NMR spectrum is recorded. The sample tube, with plastic cap removed, is clamped in front of a vertical 15-W fluorescent light, and tube and lamp are loosely wrapped with aluminum foil to increase reflection of light to the sample. A control sample, with lipid but no photosensitizer, is treated in similar manner. A third sample may be prepared containing photosensitizer and a few drops of vitamin E (α-tocopherol acetate) from a gelcap. The samples are illuminated for 10–24 h. The 1H NMR spectra are recorded. The samples may be put back in front of the light if the effects of longer light exposure are to be studied, or they may be capped and stored in the dark for a few days if it is not possible to record the spectra immediately. (Note that the color of the porphyrin generally bleaches to a yellowish brown). Alternative method: Several drops of lipid are placed on a watch glass and a few drops of acetone solution of TPP are added. (Acetone will quickly evaporate, leaving a solution of lipid and TPP.) The watch glass is placed on a lightbox or under a 15-W fluorescent lamp positioned horizontally on the bench. A control sample is placed next to the sample and irradiated for 10–24 h. A few drops of the product mixture are dissolved in a suitable solvent (CCl4, deuterated chloroform, or deuterated acetone may be used, with TMS) and the 1 H NMR spectra are measured.
Quantitative Determination of Peroxidation The volume of oxygen absorbed from the air can be measured by attaching the NMR tube with sample to a gas measuring apparatus via Tygon tubing as shown in Figure 3. The initial levels of water in the manometer should be set at least 10 mL below the uppermost calibration mark
on the buret. After irradiation the water is leveled again and the volume of oxygen absorbed can be determined. For 50 µL of methyl linoleate 3–6 mL of oxygen may be absorbed during 24 h of irradiation. The gas laws are applied to find the moles of oxygen that reacted. Results and Discussion Typical student spectra of a control and sample in deuterated acetone are shown in Figures 4 and 5. The singlet from the methyl ester group, at 3.6 ppm, is unchanged by reaction. There is a symmetrical, triplet-like signal due to alkene protons at 5.1–5.4 ppm in methyl linoleate; this changes to a broader multiplet as a mixture of conjugated and nonconjugated hydroperoxy products is formed (Fig. 5). Figure 5 also shows signals in the 4.1–4.3 ppm region that are assigned to protons on the carbon with the hydroperoxy group (4), and a signal at 10.4 ppm, which is assigned to the hydroperoxy group. Integration shows loss in intensity of the alkene protons; the ratio of the integration of alkene protons to methyl protons changes from the initial 4:3 (1.33) to approximately 1.0 after 24 h, which is consistent with formation of cyclic peroxides (6) or possibly other reactions such as dimerization. Comparison of the control and sample with photosensitizer shows that although there is a small peak at 10.3 ppm, evidence of some autoxidation, the changes such as the decrease in integration ratio (alkene:methyl protons) are significantly less. Also, a sample with photosensitizer and vitamin E shows less evidence of peroxidation than the sample with photosensitizer but without vitamin E. Conclusion These experiments demonstrate to students that lipid peroxidation occurs more rapidly when a photosensitizer is present, and also show the protective effect of the antioxidant vitamin E. By using 1H NMR spectra to monitor the reaction there is direct evidence of the structural changes that occur as a result of peroxidation. Notes 1. A dark control, a mixture of lipid and TPP in deuterated acetone kept in the dark for 24 h, shows no evidence of reaction. 2. When carbon tetrachloride is used to dissolve samples from the watch glass, no signal for the hydroperoxy group could be detected in scans down to 12 ppm. 3. Natural polyunsaturated lipids, such as corn and soybean oil, also contain vitamin E. Experiments conducted with these substances are very much slower than the experiment with pure methyl linoleate. Olive oil, however, showed marked degradation over a 3-day period. 4. The concentration of photosensitizer added is below the detection limit of 60-MHz NMR. Vitamin E signals may be seen, but do not interfere with the alkene region.
Literature Cited
Figure 3. NMR tube with sample attached to gas buret to find volume of oxygen absorbed, shown after irradiation.
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1. Simic, M. G.; Karel, M. Autoxidation in Food and Biological Systems; Plenum: New York, 1980. 2. Frimer, A. A. In Chemistry of Peroxides; Patai, S., Ed.; Wiley: New York, 1983; Chapter 7. 3. Girotti, A. W. Photochem. Photobiol. 1990, 51, 497–509. 4. Hall, G. E.; Roberts D. G. J. Chem. Soc. B 1966, 1109–1112. 5. Chan, H. W.-S. J. Am. Oil Chem. Soc. 1977, 54, 100–104. 6. Chacon, J. N.; Jamieson, G. R.; Sinclair, R. S. Chem. Phys. Lipids 1987, 43, 81–99. 7. Ohtani, B.; Nishida, M.; Nishimoto, S.-I.; Kagiya, T.
Journal of Chemical Education • Vol. 74 No. 12 December 1997
In the Laboratory
Figure 4. 1H NMR (60 MHz) spectrum of methyl linoleate in deuterated acetone, after 24 hr light exposure (control).
Figure 5. 1H NMR (60 MHz) spectrum of product mixture after 24-h photosensitized peroxidation (sample).
Photochem. Photobiol. 1986, 44, 725–732. 8. Sorato, Y.; Takahama, U.; Kimura, M. Biochim. Biophys. Acta 1984, 799, 313–317. 9. Valenzuela, A. Life Sci. 1991, 48, 301–309. 10. Heath, R. L.; Packer, L. Arch. Biochem. Biophys. 1968, 125, 189–198. 11. Tanielian, C.; Mechin, R. Photochem. Photobiol. 1994, 59, 263–268.
12. Terao, J.; Kawakatsu, M.; Matsushita, S. Lipids 1981, 16, 427–432. 13. Neff, W. E.; Frankel, E. N.; Miyashita, K. Lipids 1990, 25, 33–39. 14. Claxson, A. W. D.; Hawkes, G. E.; Richardson, D. P.; Naughton, D. P.; Hayward, R. M.; Chandler, C. L.; Atherton, M.; Lynch, E. J.; Grootveld, M. C. FEBS Lett. 1994, 355, 81– 90.
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