Abstract
We have developed an assay for the isolation and quantitation by gas chromatography-mass spectrometry (GC-MS) of free 9- and 13-hydroxyoctadecadienoic acid (9-HODE, 13-HODE) in the mammary glands of female mice. Internal standards consisting of 18O2-labeled analogs of 9- and 13-HODE are added to pulverized frozen tissue prior to extraction with ethanol. Nonlipid materials are removed in a chloroform/methanol/water extraction step. The remaining lipid material is methylated with ethereal diazomethane, and much of the nonoxygenated fatty acid methyl esters are removed via silica solid-phase extraction. Samples are either further derivatized with bis(trimethylsilyl)trifluoroacetamide to form the trimethylsilyl ethers for quantitative analysis by GC-MS or are analyzed as the methyl esters by chiral high-performance liquid chromatography to determine the enantiomeric distribution of the 9- and 13-HODE. The extraction and quantitation protocol was applied to the analysis of mammary glands for free 9- and 13-HODE from mice fed isocaloric diets containing 20% corn oil, 5% corn oil, or 20% beef tallow. Chiral analysis of the products showed higher production of 13(S)-HODE relative to 13(R)-HODE; the enantiomeric excess is most likely due to enzymatic production of 13-HODE superimposed on a background of autoxidative production of 13(R)-plus 9(S)- and 9(R)-HODE. In addition, the effect of sample handling and storage conditions on the formation of 9- and 13-HODE in the samples was assessed by exposing aliquots of a common pool of rat mammary gland tissue to specified conditions prior to analysis. This methodology will be important during investigations of the contribution of linoleate oxidation products to the enhancement of mammary tumorigenesis by dietary fat.
Similar content being viewed by others
Abbreviations
- ANOVA:
-
analysis of variance
- BHT:
-
butylated hydroxytoluene
- BSTFA:
-
bis(trimethylsilyl)trifluoroacetamide
- GC-MS:
-
gas chromatography-mass spectrometry
- 9-HODE:
-
9-hydroxyoctadecadienoic acid
- 13-HODE:
-
13-hydroxyoctadecadienoic acid
- HPLC:
-
high-performance liquid chromatography
- ME:
-
methyl ester
- TMCS:
-
trimethylchlorosilane
- TMS:
-
trimethylsilyl
References
Welsch, C.W. (1995) Review of the Effects of Dietary Fat on Experimental Mammary Gland Tumorigenesis: Role of Lipid Peroxidation, Free Radical Biol. Med. 18, 757–773.
Rose, D.P., Hatala, M.A., Connolly, J.M., and Rayburn, J. (1993) Effects of Diets Containing Different Levels of Linoleic Acid on Human Breast Cancer Growth and Metastasis in Nude Mice, Cancer Res. 53, 4686–4690.
Bull, A.W., Nigro, N.D., Golembieski, W.A., Crissman, J.D., and Marnett, L.J. (1984) In Vivo Stimulation of DNA Synthesis and Induction of Ornithine Decarboxylase in Rat Colon by Fatty Acid Hydroperoxides, Autoxidation Products of Unsaturated Fatty Acids, Cancer Res. 44, 4924–4928.
Bull, A.W., Nigro, N.D., and Marnett, L.J. (1988) Structural Requirements for Stimulation of Colonic Cell Proliferation by Oxidized Fatty Acids, Cancer Res. 48, 1771–1776.
Glasgow, W.C., and Eling, T.E. (1990) Epidermal Growth Factor Stimulates Linoleic Acid Metabolism in Balb/C 3T3 Fibroblasts, Mol. Pharmacol. 38, 503–510.
Glasgow, W.C., Afshari, C.A., Barrett, J.C., and Eling, T.E. (1992) Modulation of the Epidermal Growth Factor Mitogenic Response by Metabolites of Linoleic and Arachidonic Acid in Syrian Hamster Embryo Fibroblasts. Differential Effects in Tumor Suppressor Gene (+) and (−) Phenotypes, J. Biol. Chem. 267, 10771–10779.
Yurek, E.M. (1990) A Selective Extraction Technique for Hydroxylated Fatty Acids in Mammary Tissue Based in Analysis by Gas Chromatography/Mass Spectrometry, Master’s Thesis, Michigan State University, East Lansing, pp. 9–28.
Westcott, J.Y., Clay, K.L., and Murphy, R.C., Preparation of Oxygen-18-labeled Lipoxygenase Metabolites of Arachidonic Acid, Biomed. Mass. Spectrom. 12, 714–718.
Lehmann, W.D., Stephan, M., and Fürstenburger, G. (1991) Profiling Assay for Lipoxygenase Products of Linoleic and Arachidonic Acid by Gas Chromatography-Mass Spectrometry, Anal. Biochem. 204, 158–170.
Graff, G., Anderson, L.A., and Jaques, L.W. (1990) Preparation and Purification of Soybean Lipoxygenase-Derived Hydroperoxy and Hydroxy Fatty Acids and Determination of Molar Absorptivities of Hydroxy Fatty Acids, Anal. Biochem. 188, 38–47.
Welsch, C.W., DeHooq, J.V., O’Conner, D.H., and Sheffield, L.G. (1985) Influence of Dietary Fat Levels on Development and Hormone Responsiveness of the Mouse Mammary Gland, Cancer Res. 45, 6147–6154.
Bligh, E.G., and Dyer, W.J. (1959) A Rapid Method of Total Lipid Extraction and Purification, Can. J. Biochem. Physiol. 37, 911–917.
van Kuijk, F.J.G., Thomas, D.W., Stephens, R.J., and Dratz, E.A. (1985) Gas Chromatography-Mass Spectrometry Method for Determination of Phospholipid Peroxides; I. Transesterification to Form Methyl Esters, Free Radical Biol. Med. 1, 215–225.
van Kuijk, F.J.G., Thomas, D.W., Stephens, R.J., and Dratz, E.A. (1985) Gas Chromatography-Mass Spectrometry Method for Determination of Phospholipid Peroxides; II. Transesterification to Form Pentafluorobenzyl Esters and Detection with Picogram Sensitivity, Free Radical Biol. Med. 1, 387–393.
Guido, D.M., McKenna, R., and Mathews, W.R. (1993) Quantitation of Hydroperoxyeicosatetraenoic Acids and Hydroxyeicosatetraenoic Acids as Indicators of Lipid Peroxidation Using Gas Chromatography-Mass Spectrometry, Anal. Biochem. 209, 123–129.
Watson, J.T. (1985) Introduction to Mass Spectrometry, 2nd edn., pp. 59–72, Raven Press, New York.
Hubbard, W.C., Hough, A.J., Jr., Brash, A.R., Watson, J.T., and Oates, J.A. (1980) Metabolism of Linoleic and Arachidonic Acids in VX2 Carcinoma Tissue: Identification of Monohydroxy Octadecadienoic Acids and Monohydroxy Eicosatetraenoic Acids, Prostaglandins 20, 431–447.
Funk, M.O., Isaac, R., and Porter, N.A. (1976) Preparation and Purification of Lipid Hydroperoxides from Arachidonic and γ-Linoleic Acids, Lipids 11, 113–117.
Kühn, H., Wiesner, R., Lankin, V.Z., Nekrasov, A., Alder, L., and Schewe, T. (1987) Analysis of the Stereochemistry of Lipoxygenase-Derived Hydroperoxy Polyenoic Fatty Acids by Means of Chiral-Phase High-Pressure Liquid Chromatography, Anal. Biochem. 160, 24–34.
Glasgow, W.C., and Eling, T.E. (1994) Structure-Activity Relationship for Potentiation of EGF-Dependent Mitogenesis by Oxygenated Metabolites of Linoleic Acid, Arch. Biochem. Biophys. 311, 286–292.
Baer, A.N., Costello, P.B., and Green, F.A. (1990) Free and Esterified 13(R,S)-Hydroxyoctadecadienoic Acids: Principal Oxygenase Products in Psoriatic Skin Scales, J. Lipid Res. 31, 125–130.
Baer, A.N., Costello, P.B., and Green, F.A. (1991) Stereospecificity of the Products of Fatty Acid Oxygenases Derived from Psoriatic Scales, J. Lipid Res. 32, 341–347.
Author information
Authors and Affiliations
About this article
Cite this article
Johnson, J.A., Blackburn, M.L., Bull, A.W. et al. Separation and quantitation of linoleic acid oxidation products in mammary gland tissue from mice fed low- and high-fat diets. Lipids 32, 369–375 (1997). https://doi.org/10.1007/s11745-997-0047-7
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s11745-997-0047-7