Abstract
Soybean lipoxygenase (LOX; EC 1.12.11.12) catalyzes the oxygenation of polyunsaturated fatty acids, acylglycerols and phosphoglycerols, producing a regio-and enantiospecific hydroperoxide product. The goal of this work was to measure the relative rate of LOX-catalyzed oxidation of mixtures of lipids containing linoleate, using high-performance liquid chromatography (HPLC) and a light-scattering detector (LSD). Previous literature sugested that reversed-phase HPLC with silicabased columns could be used for the separation of individual fatty acids, acylglycerols, phosphoglycerides and their oxidation products. However, these columns produced ineffective separations of phosphoglycerides unless choline chloride and a strong base, such as KOH, are present in the mobile phase. Such modifiers precluded the use of the LSD. It was found that a reversed-phase column based upon an organic polymer support, rather than on silica, was able to separate these mixtures with a ternary solvent gradient of methanol/water/acetonitrile without the need for the addition of modifiers. The oxidation time course of a mixture of linoleic acid, trilinolein and 1-linoleoyl-2-stearoyl-sn-glycero-3-phosphocholine was followed using the developed HPLC method. The results showed that trilinolein and phosphatidylcholine reacted at one-tenth the rate of linoleic acid. The diacylglycerol, 1,3-dilinolein, was oxidized at a rate that was approximately 40% that of linoleic acid, with the formation of mono-and dihydroperoxides as well as other unidentified products.
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Abbreviations
- 1,3-DL:
-
1,3-dilinolein
- DO:
-
1,3-diolein
- HPLC:
-
high-performance liquid chromatography
- LA:
-
linoleic acid
- LSD:
-
light-scattering detector
- IL-2S-PC:
-
1-linoleoyl-2-stearoyl-sn-glycero-3-phosphocholine
- LOX:
-
lipoxygenase
- NP:
-
normal phase
- 1P-2L1-PE:
-
1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine
- RP:
-
reversed phase
- TL:
-
trilinolein
- UV:
-
ultraviolet
References
Hamberg, M., and Samuelsson, B., (1965)Biochem. Biophys. Res. Commun. 21, 431–536.
Holman, R.T., Egwim, P.O., and Christie, W.W. (1969)J. Biol. Chem. 244, 1149–1151.
Hamberg, M. (1971)Analytical Biochemistry 43, 515–526.
Parra-Diaz, D., Brower, D.P., Medina, M.B., and Piazza, G.J. (1993)Biotechnol. Appl. Biochem. 18, 359–367.
Piazza, G.J., Brower, D.P., and Parra-Diaz, D. (1994)Biotechnol. Appl. Biochem. 19, 243–252.
Koch, R.B., Stern B., and Ferrari, C.G. (1969)Arch. Biochem. Biophys. 78, 165–179.
Guss, P.L., Richardson, T., and Stahmann, M.A. (1968)J. Am. Chem. Soc. 45, 272–276.
Christofer, J.P., Pistorius, E., and Axelrod, B. (1970)Biochem. Biophys. Acta 198, 12–19.
Eskola, J., and Laakso, S. (1983)Biochem. Biophys. Acta 751, 305–311.
Brash, A.R., Ingram, C.D., and Harris, T.M. (1987)Biochemistry 26, 5465–5471.
Schellenberger, V., Siegel, R.A., Rutter, W. (1993)Biochemistry 32, 4344–4348.
Christie, W.W. (1985)Z. Lebensm.-Unters Forsch. 181, 171–182.
Antonoppoulou, S., Andrikopoulos, N.K., and Demopoulos, C.A. (1994)J. Liquid Chromatogr. 17, 633–648.
Patton, G.M., Fasulo, J.M., and Robins, S.J. (1982)J. Lipid. Res. 23, 190–196.
Therond, P., Couturier, M., Demelier, J.F., and Lemonnier, F. (1993)Lipids 28, 245–249.
van Kuijk, F.J.G.M., Thomas, D.W., Stephens, R.J., and Dratz, E.A. (1985)J. Free Radicals Biol, Med. 1, 215–225.
Wehrli, A., Hildenbrand, J.C., Keller, H.P., and Stampfli, R. (1978)J. Chromatogr. 149, 199–210.
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Nuñez, A., Piazza, G.J. Analysis of lipoxygenase kinetics by high-performance liquid chromatography with a polymer column. Lipids 30, 129–133 (1995). https://doi.org/10.1007/BF02538265
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DOI: https://doi.org/10.1007/BF02538265