Development of a P-32-postlabeling method for the detection of 1,N-2-propanodeoxyguanosine adducts of trans-4-hydroxy-2-nonenal in vivo

A P-32-postlabeling method was developed for the sensitive detection of 1,N -2-propanodeoxyguanosine adducts of the lipid peroxidation product trans-4- hydroxy-2-nonenal in vivo. The method development was based on the chemical ly synthesized HNE - 1 ,N-2-propanodeoxyguanosine adduct standard, which wa s characterized by NMR and mass spectra. The adducts were enriched by nucle ase P1. They were subsequently reacted with [gamma-P-32]ATP to give the res pective 3'-5'-bisphosphates, which were two-directionally separated on PEI- cellulose TLC and quantitated by autoradiography. The medium labeling effic iency for the mixture of the two pairs of diastereomers was 27%, and the re covery of spiked amounts of adduct standard in the enzymatical procedure wa s about 80%. The method is applicable for the separation and quantitation o f HNE-dGp-propano adducts in vivo. It was applied to DNA from colon and bra in tissue of untreated Fischer 344 rats and humans. The determination of th e limit of quantitation in DNA from rat colon by spiking of adduct standard revealed a sensitivity of <21 adducts/10(9) nucleotides. The analytical qu antitation of 4-HNE-dGp-propano adducts resulted in adduct-levels per 10(9) normal nucleotides +/- the standard deviation of 223.32 +/- 79.84 in rat c olon tissue, 90.37 +/- 11.94 in rat brain tissue, 378.44 +/- 52.42 in human colon tissue, and 185.15 +/- 6.48 in human brain tissue. The results clear ly demonstrate the applicability of this method for the sensitive detection of endogenously formed 1,N-2-propanodeoxyguanosine adducts of trans-4-hydr oxy-2-nonenal, a specific marker for the lipid peroxidation process.