Ascorbic acid assays of individual neurons and neuronal tissues using capillary electrophoresis with laser-induced fluorescence detection.
Ascorbic acid is an important cellular metabolite involved in many biochemical pathways. A method to quantitate ascorbic acid and dehydroascorbic acid in individual neurons and neuronal tissues is described with detection limits of 320 pM (430 zmol). The method uses microvial sampling, derivatization with 4,5-dimethyl-1,2-phenylenediamine, capillary electrophoresis separation, and laser-induced fluorescence detection and quantifies the ascorbic acid and dehydroascorbic acid levels with less than a 15-min total analysis time including sample preparation and derivatization. Ascorbic acid and dehydroascorbic acid levels are measured using functionally characterized and identified neurons of Aplysia californica, Pleurobranchaea californica, and Lymnaea stagnalis -three well-recognized models in cellular and system neuroscience. Multiple assays of a particular identified neuron (e.g., metacerebral cells from Aplysia) show a high level of reproducibility, while endogenous intracellular concentrations of ascorbate are neuron-specific. Ascorbic acid concentrations in the neurons studied range from 0.19 to 6.2 mM for Aplysia and 0.12 to 0.22 mM for Lymnaea. In contrast, concentrations of ascorbic acid observed in heterogeneous tissues such as ganglia (with connective tissues, glia, blood vessels, neuropile, and areas with intercellular spaces), 4-190 microM, are significantly lower than the single-cell values.
Kim
Won-Suk
WS
Department of Chemistry and the Beckman Institute, University of Illinois, Urbana 61801, USA.
Dahlgren
Robin L
RL
Moroz
Leonid L
LL
Sweedler
Jonathan V
JV
D000818:Animals; D001048:Aplysia / Q000737:chemistry / Q000166:cytology; D001205:Ascorbic Acid / Q000032:analysis* / Q000378:metabolism; D003683:Dehydroascorbic Acid / Q000032:analysis / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods; D007834:Lasers; D009474:Neurons / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods
D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid