Detection and measurement by high-performance liquid chromatography of malondialdehyde crosslinks in DNA

Malondialdehyde, which is generated by lipid peroxidation, can form DNA-protein and/or interstrand DNA crosslinks. The biological consequences of inaccurate repair of these crosslinks may be severe. The expected levels of crosslinking of DNA in vivo are low, and an extremely sensitive method must be used for their detection and measurement. Because both types of crosslinks may contain cytosine, the cytosine residues of DNA were labeled in vitro with 125I. The iodinated DNA was treated with Penicillium nuclease P1 at pH 6 and with alkaline phosphatase at pH 9, and the nucleosidic compounds were analyzed by high-performance liquid chromatography. The optimum conditions for measurement of the crosslinks on Ultrasphere ODS or Zorbax ODS columns were 50 mM ammonium phosphate buffer, pH 7, that contained 2% methanol and 5 mM tetra-t-butylammonium phosphate. Both DNA-protein and interstrand DNA crosslinks were measurable simultaneously. The method was quantitative, reproducible, and able to detect crosslinked adducts at subpicomole levels, so that as few as two crosslinks per 10(6) base pairs were detectable.