Conclusion

The method described here is the only one currently available for the detection of isomeric HNE adducts in tissue DNA. Because it is a multiple-step method of superior sensitivity, caution must be taken to avoid contamination. Some of the precautions are as follows: (1) Use the designated HPLC unit pipettes and pipette tips for in vivo samples. Pipettes should be cleaned once a week by soaking in soapy water overnight, and rinsing thoroughly with water; (2) dispense the enzyme solutions to several microcentrifuge tubes to minimize the freeze-thaw process and possible cross-contamination; (3) store the kinase buffer at —80° for no more than 6 months; and (4) wash all TLC sheets by developing in water for 24 hr, air dry, and keep frozen at —20° before use. Although this method is routinely used in our laboratory, it still can be refined and improved. To perform this assay for the first time in the laboratory, it is highly desirable to initially use the synthetic HNE-dG standards before using DNA samples. The detection and identification capability of this assay for the isomeric HNE-dG adducts is clearly a unique strength. Using this assay, our studies have demonstrated its potential application for analyzing HNE-dG adducts in tissue as markers of endogenous DNA damage by lipid peroxidation. These studies will eventually help us understand the molecular mechanisms of chronic human diseases, including cancers, cardiovascular diseases, and neurodegenerative brain disorders.

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