Determination Of Amino Acid Neurotransmitters By High Performance Capillary Electrophoresis(HPCE) In Hippocampus Of Rats And Cerebrospinal Fluid Of Epilepsy Patients

Capillary electrophoresis (CE) is a relatively new separation technique compared to the well-known chromatographic procedures, such as HPLC and gas chromatography separation of a complex array of large and small molecules, including proteins, amino acids, nucleotides, varieties of ions. The technology of CE can be segmented into several separation techniques,including capillary zone electrophoresis(CZE), capillary gel electrophoresis(CGE),micellar electrokinetic capillary chromatography(MECC), isoelectric focusing(IEF),isotachophoresis(ITP). The forms which we used often are capillary zone electrophoresis (CZE). Fundamental to CZE are homogeneity of the buffer solution and constant field strength throughout the length of the capillary. In this article, we establish a rapid assay of determining amino acid neurotransmitters in brains of rats by capillary electrophoresis with laser-induced fluorescence . HPCE has many advantages such as rapidity, simplicity, accuracy and sensitivity. The method can be used in clinical and scientific research.PartⅠEstablishment a method for separating neurotransmitters by capillary electrophoresis with laser-induced fluorescence detectionGLU,ASP,GABA,GLY were separated together by capillary electrophoresis with laser-induced fluorescence detection. The running buffer was 100 mmol/L boric acid-Tris contained 1% isopropyl alcohol, pH 10.3.The voltage of 18 KV was applied. The level of four amino acids were measured by laser induced fluorescence detector. The known weight's mice brains were homogenated with 0.1mol/L perchloric acid flowed the ratio 10ml perchloric acid to 1g brain mass. After homogenated,the fluid were centrifuged and the supernatant fluid were stored at -70℃until detected. The complete separation of 4 amino acids was achieved within 15 min. The level of four amino acids were 0.67±0.69,2.65±0.19,2.31±0.21,2.86±0.38umol/g respectively. The linearity of the assay was from 1.0μmol/L to 100.0μmol/L .The minimal detection limit were10 ,23 ,18 ,15 nmol/L respectively . For those amino acids, good precisions were obtained between 4.44%～9.72% for the Within-run precisions and 6.96%～10.50% for the Between-run precisions . The recovery of amino acids were not less than 88.1%. This method will make the research of physio-pathology of neurotransmitter and detection of concentration of pharmaceutic neurotransmitter exactly and quickly.Part II Application of capillary zone electrophoresis on Cerebrospinal fluid of epilepsy patients45 cases of cerebrospinal fluid specimens (SCF) were collected. They were divided into control group, partial complex group , general epilepsy group .GLU,ASP,GABA,GLY are 34.34±4.65,50.28±6.85,48.32±5.24,25.72±3.56umol/L respectively in control group, GLU,ASP,GABA,GLY are 54.64±7.80,69.65±8.32,52.54±6.28,38.22±5.34umol/L in general epilepsy group,GLU,ASP,GABA,GLY are 78.85±9.55,91.07±11.23,73.65±9.64,50.33±8. 98 umol/L in partial complex group. The levels of GLU,GLY,ASP in CSF of general epilepsy patients were significantly higher than in control group(P<0.05), but the level of GABA was not significantly differen(tP>0.05); the concentrations of GLU,GLY,ASP and GABA in CSF of partial complex epilepsy were obviously higher than in control group(P<0.05). The levels of GABA in CSF of general epilepsy patients were significantly higher than in control group and general epilepsy patients (P<0.05).The method to detect amino acid in cerebrospinal fluid is convenient by capillary electrophoresis using laser-induced fluorescence and can be used to epilepsy disease research. SummaryCompared with traditional High Performance Liquid Chromatagraghy (HPLC) ,Capillary electrophoresis is a new analytical technique. It offers advantages for amino acids analysis including high resolution, rapid analysis, good reprodution, automation and low consumption of sample. The approach greatly improved the speed, resolution and sensitivity .The result indicated that the method was an effective method for clinical and scientific research .