Methods are being developed and tested to detect oxidative damage to DNA in non-dividing cells in order to determine whether hydroxyl radicals generated either by redox cycling a toxin utilizing endogenous enzymes, or as a result of metabolism which has been activated by a toxin, may be involved in the pathogenesis of neurodegenerative disorders. Oxidative damage to neuronal DNA could result in impaired neuronal function if the damage remained unrepaired and accumulated. In order to measure DNA damage , either neuronal or mitochondrial, gas chromatographic-mass spectrometric methods are being improved for the detection of thymine glycol or thymidine glycol, and thymidine. A high sensitivity negative ionization mass spectrometric assay of 2-methylglycerate, a hydrolysis- reduction product from thymidine has developed to work with 10 fg quantities of DNA, a 20-fold improvement with respect to an electron ionization assay. Efficient pentafluorobenzyl esterification of 2- methylglycerate was possible using tetrabutylammonium assisted anhydrous derivatization. Platelet activating factor has been measured in cell and animal models of neuropathology accompanying inflammation. The gerbil cerebral ischemia model is being used for in vivo assessment. Collaborative studies on the identification of unknown biologically active materials are in progress - a pituitary derived cytotrophic factor, and a brain or plasma derived benzodiazepine ligand binding substance. In both studies, on-line chromatography and various mass spectrometric techniques are being used to identify materials from complex mixtures that have extensively chromatographed, but still contain interfering substances.
