1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is an exogenous (synthetic) neurotoxin inducing Parkinsonism in humans, non-human primates, and rats. Salsolinol (6,7-dihydroxy-1-methyl-1, 2, 3, 4 -tetrahydroisoquinoline, Sal) is an MPTP-like neurotoxin. Some of its phase I metabolites, e.g. N-methyl- (R)-Sal, have been shown to target and injure particularly dopaminergic nigrostriatal neurons inducing Parkinsonism. Unfortunately, Sal can be formed in vivo from the condensation of dopamine (a neurotransmitter in the brain) and acetaldehyde (a metabolite of alcohol). Very interestingly, the two enantiomers of N-methyl-Sal exhibit distinct neurotoxicological properties. N-methyl-(R)-Sal induces Parkinsonism in rats, but N-methyl-(S)-Sal does not. However, the stereochemical aspects of in-vivo formation and metabolism of these neurotoxic enantiomers remain largely unknown. For example, literature data on the stereospecific occurrence of Sal in physiological fluids are inconsistent. The goal of this research is to identify the biosynthetic and metabolic pathways of Sal enantiomers. To this end, the development of suitable analytical methodology for simultaneous quantification of the enantiomers of Sal and its phase I metabolites is needed. We are proposing to develop a chiral analytical method based on capillary HPLC /tandem mass spectrometry for this purpose. After the analytical methodology is in place, studies will be carried out: 1) to determine the enantiomeric compositions of endogenous Sal and its metabolites in rat brain and physiological fluids;2) to investigate in vivo formation of Sal in rat brain using microdialysis after administering 13C labeled acetaldehyde and pyruvic acid to rats;3) to profile the phase I metabolites of Sal and N-methyl-Sal using 13C labeled Sal or N-methyl-Sal enantiomers (e.g. [1 ,m-13C2]-salsolinol);and 4) to assess the neurotoxicity of enantiomeric Sal and its major phase I metabolites including N-methyl-Sal and N-methyl-4-hydroxyl-Sal in PC-12 and SH-SY5Y cells. The hypothesis is that racemization of the more neurotoxic (R)-enantiomers of endogenous Sal and its metabolites is a major pathway leading to detoxification in a healthy nervous system. By using the improved methodology for chiral analysis in combination with in vivo microdialysis and stable isotope labeling techniques, the research will contribute significantly to our knowledge of in vivo formation and metabolism of these neurotoxins involved in the pathogenesis of certain neurodegenerative disorders such as Parkinson's disease. From these studies, significant gains will also be made in understanding the stereochemical aspects of neurotoxicity in general.
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