High-performance liquid chromatography with electrochemical detection for determination of the major malondialdehyde-guanine adduct.
A method is described for quantitative analysis of the pyrimidopurinone adduct (M1G) arising from reaction of malondialdehyde (MDA) with DNA. DNA samples treated with MDA are reduced with sodium borohydride and hydrolyzed with 0.1 N HCl. The bases released are isolated by solid-phase extraction and analyzed by high-performance liquid chromatography with electrochemical detection. 5,6-Dihydro-M1G (H2M1G) is detected with a glassy carbon electrode at an applied potential of 700 mV vs Ag/AgCl. At this potential, interference from normal deoxynucleoside bases is low. The limit of detection of H2M1G eluted from an HPLC column is 100-200 fmol. The method described should be useful for quantitation of M1G in a variety of DNA samples and biological fluids.
Goda
Y
Y
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Marnett
L J
LJ
D001894:Borohydrides / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis*; D004563:Electrochemistry; D006147:Guanine / Q000032:analysis*; D006851:Hydrochloric Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008315:Malondialdehyde / Q000032:analysis* / Q000737:chemistry; D010084:Oxidation-Reduction
D001894:Borohydrides; D008315:Malondialdehyde; D006147:Guanine; C025364:sodium borohydride; D004247:DNA; D006851:Hydrochloric Acid