Support is being requested for continuation of studies pertaining to the relationship between certain in vivo formed neurotoxic agents and diabetic neuropathy. Previous studies of this laboratory support the hypothesis that these toxic metabolites are the underlying factor in the development of axonal disease associated with diabetes. Unsaturated aldehydes and related oxygenated compounds are products of the lipid peroxidation process. Their formation will be followed in rat liver preparations under various dietary circumstances. The state-of-the-art sample preconcentration techniques and analytical separations will be developed with a particular emphasis on chemical instability of these reactive compounds. Supercritical fluid extraction, capillary supercritical fluid chromatography, microcolumn liquid chromatography and capillary gas chromatography/mass spectrometry will primarily be utilized. Alternative metabolic sources of neurotoxins to be investigated will include arachidonic acid and plasmalogens. The metabolic differences between normal and streptozotocin-diabetic rats will be studied. The previous findings of this laboratory that the mutant diabetic db/db mice produce the neurotoxic substances will be followed with emphasis on a possible role of dietary vitamin E in reducing disease complications. The dynamics of excretion of the urinary carbonyl compounds will be followed in long term experiments by chromatographic techniques. Further experiments will be centered around the previously observed inhibition of key glycolytic enzymes, glyceraldehyde -3-phosphate dehydrogenase and phosphofructokinase by various aldehydic metabolites, which are presumed to be a major complication of proper axonal transport of biochemicals. Enzyme kinetic measurements will be supplemented by circular dichroism measurements, size exclusion chromatography and capillary electrophoresis to elucidate chemical changes caused by the aldehydic inhibitors to glycolytic enzymes and certain other model proteins. Following incubation of proteins with these putative neurotoxins, enzymatic digestions will be performed, and the corresponding peptides maps will be developed with the aid of capillary electrophoresis and microcolumn liquid chromatography. These unique separation techniques will further be combined with tandem mass spectrometry (MS/MS) to locate the sites of damage to protein molecules.
