In vivo monitoring of amino acids by direct sampling of brain extracellular fluid at ultralow flow rates and capillary electrophoresis.
Extracellular levels of glutamate (GLU), aspartate (ASP), glycine (GLY), phosphoethanolamine (PEA), and gamma-aminobutyric acid (GABA) were measured in the striatum of anesthetized rats using a novel sampling approach in which extracellular fluid (ECF) was removed at 1-50 nl/min using a fused silica capillary tube with 18-40 microm inner diameter and a outer diameter of 90 microm. The samples of ECF were analyzed by capillary electrophoresis with laser-induced fluorescence detection. Basal levels for GABA, GLY, and GLU measured using direct sampling at 1 nl/min were 270 +/- 40, 4950 +/- 1100, and 1760 +/- 150 nM, respectively in good agreement with the values obtained using microdialysis sampling calibrated by the low-flow rate method. ASP levels were approximately four-fold higher in directly sampled fluid than in dialysate. At higher direct sampling flow rates (10-50 nl/min), detected levels of the amino acids were lower by 70-90% indicating depletion of analyte under these conditions. PEA, an indicator of membrane disruption, was 5.5-fold higher in dialysate than in directly sampled ECF indicating greater tissue damage associated with microdialysis. In addition to the basal measurements, the direct sampling technique was applied to monitoring concentration changes of GLU and ASP in the striatum with better than 90 s temporal resolution after perfusion of either 120 mM K(+) or 400 microM L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) through a microdialysis probe immediately adjacent to the direct sampling capillary. Levels of GLU and ASP increased 615 +/- 95 and 542 +/- 96%, respectively (n=4) upon addition of 120 mM K(+) to the perfusate and 622 +/- 234 and 672 +/- 218% (n=5) for PDC. It is concluded that direct sampling at low-flow rates allows determination of extracellular levels of the amino acids with spatial resolution that is at least 500-fold better than microdialysis.
Kennedy
Robert T
RT
Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, FL 32611-7200, USA. rtkenn@chem.ufl.edu
Thompson
Jonathan E
JE
Vickroy
Thomas W
TW
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001921:Brain / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004983:Ethanolamines / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D005453:Fluorescence; D018698:Glutamic Acid / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D007834:Lasers; D017551:Microdialysis / Q000295:instrumentation* / Q000379:methods*; D051381:Rats; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D000596:Amino Acids; D004983:Ethanolamines; D001224:Aspartic Acid; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid; C005448:phosphorylethanolamine; D005998:Glycine