Gas chromatographic-mass spectrometric method for the assessment of oxidative damage to double-stranded dna by quantification of thymine glycol residues.
A technique for the measurement of thymine glycol at parts per million concentrations in double-stranded polymeric DNA is described. The procedure utilizes base to ring-open DNA-bound thymine glycol in the presence of monomeric [(2)H4]thymine glycol as an internal standard, followed by reduction, solvolytic cleavage, and quantification of the characteristic methyl-2-methylglycerate released from polymeric DNA. Methyl-2-methyl-glycerate is derivatized to form the di-tert-butyldimethylsilyl [(TBDMS)2] ether to enhance its gas chromatographic properties and electron ionization detection. This assay was tested by measuring thymine gIyco1 levels in native, undamaged DNA (not purposefully oxidized). The measured quantities of thymine glycol are proportional to the amount of DNA analyzed. Components of DNA not containing oxidizable thymine do not contribute to the measured signal from methyl-2-methylglycerate-(TBDMS)2. These results indicate that there is approximately one thymine glycol per lo6 bases in undamaged DNA and that this value increases with storage of DNA in refrigerated aqueous solutions.
Section on Analytical Biochemistry, Laboratory of Clinical Science, National Institute of Mental Health, Building 10, Room 3D40, NIH, 20892, Bethesda, MD.
S P
SP
Markey
C J
CJ
Markey
T C
TC
Wang
J B
JB
Rodriguez