title: Determination of lactate dehydrogenase in human erythrocytes by capillary electrophoresis with electrochemical detection.
abstract: Capillary electrophoresis (CE) was employed to analyze lactate dehydrogenase (LDH) in human erythrocytes using an amperometric detector with a carbon fiber micro-disk bundle electrode. LDH activity was measured by determining the amount of NADH generated by LDH through a enzyme-catalyzed reaction between NAD(+) and lithium lactate. The factors influencing the enzyme-catalyzed reaction, separation and detection were examined and optimized. The following conditions were suitable for the determination of LDH: running buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 7.5); separation voltage, 20.0 kV; detection potential, 1.00 V (versus saturated calomel electrode (SCE)). The conditions of enzyme-catalyzed reaction were: reaction buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 9.3); substrates, 5.0 x 10(-2)mol/l lithium lactate and 5.0 x 10(-3)mol/l NAD(+); reaction time, 10 min. The concentration limit of detection (LOD) of the method was 0.017 U/ml at a signal-to-noise (S/N) ratio of 3, which corresponded to 1.10 x 10(-10)mol/l, and the mass LOD was 2 x 10(-20)mol. The linear dynamic range was 0.039-4.65 U/ml for the injection voltage of 5.0 kV and injection time of 10s. The relative standard deviation (R.S.D.) was 0.85% for the migration time and 1.8% for the electrophoretic peak area. The method was applied to determine LDH in human erythrocytes. The recovery of the method was between 98 and 101%.
authors: School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China.
authors: Wei
authors: W
authors: Wang
authors: Xuemei
authors: X
authors: Sun
authors: Wenrui
authors: W
authors: Jin
mesh_terms: D004563:Electrochemistry / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000097:blood*; D012680:Sensitivity and Specificity
chemical_list: D007770:L-Lactate Dehydrogenase