HPLC analysis of lipid hydroperoxides and fat-soluble vitamins: Application to diabetes and obesity

Problem under investigation. Lipid peroxidation is a prominent manifestation of oxidative stress because of cytotoxic effects and possible role in pathological conditions like atherosclerosis, ischemia-reperfusion injury, drug induced liver damage and carcinogenesis. Current methods for determination of lipid peroxidation products are non-specific, or prohibitively complex. Therefore, a simple isocratic HPLC method for the determination of polyunsaturated fatty acid lipid hydroperoxides (LHP) using ultraviolet absorption detection will be developed.; Previous findings. Most attempts at LHP detection by NPLC have omitted ultraviolet absorption due to its inability to differentiate LHP from hydroxy reduction products which co-elute. These methods require electrochemical detection or post-column chemiluminescence to specifically detect the hydroperoxides.; Objective/hypothesis. Recent research has shown that LHP can be separated from their hydroxy derivatives by reverse phase HPLC. Furthermore, the four major fatty acids ie; linoleic, linolenic, arachidonic, and docosaehexanoic acid, their hydroperoxides and hydroxy derivatives were resolved on a single chromatogram. Since the primary site of oxidation is on the esterified portion of cholesterol, phospholipid or triglyceride, this HPLC technique could be applied to lipid extracts in which all fatty acids have been freed from their ester bonds. The method offers a complete "lipid peroxidation profile" in which the extent of peroxidation can be characterized.; Research design/materials/methods. Lipoprotein fractions (VLDW LDL and HDL) will be isolated by precipitation techniques and verified for purity by agarose gel electrophoresis. Lipid extracts will be prepared by a modification of the Folch extraction and by saponification. Finally, these preparations will be subjected to reversed phase HPLC and quantified in comparison to standards synthesized by enzymatic oxidation of authentic standards. The fat soluable vitamins A,E, beta-carotene, lutein, zeaxanthin and beta-cryptoxanthin will also be determined to see if their antioxidant properties relate to the level of oxidation.; Preliminary results. Preliminary results indicate that the method can resolve LHP and hydroxy derivatives at the picomole level. Furthermore, exposure of whole serum and isolated lipoprotiens to mild oxidizing conditions causes easily detected increases in peroxidation products and corresponding decreases in fat soluable vitamins. Method evaluation shows excellent linearity, recovery and reproducibility of results for both methods.; Intended methods for data analysis. Power analysis will be used to determine the number of patients samples required to generate significance. Quantitation of integrated chromatogram peaks will be performed by least squares linear regression from known standard peak areas. The results will be evaluated for statistical significance (p {dollar}<{dollar} 0.05) using Student's paired and unpaired T-tests.; Potential significance. Lipid peroxidation may be the seminal event leading to the appearance and progression of diabetic complications as well as related disorders. This method would serve as an invaluable tool for the evaluation and monitoring of oxidative stress.