Determination of lactate or oxalate using injected lactate oxidase and peroxidase by capillary electrophoresis with UV detection.
Two reactions, catalyzed by lactate oxidase (LO) and peroxidase, are initiated by a single injection of the enzymes and the substrate 2,2'-azino-bis(3-ethylene-thiazoline-6-sulfonic acid) (ABTS) into the capillary previously filled with the sample (lactate or lactate-oxalate mixture) and the run buffer containing NADH. The oxidized ABTS product upon reaction with NADH is converted to NAD(+) which is separated and detected in less than 2 min at 266 nm with a sample throughput of 7 min (including wash steps between samples). Simplex trade mark software is used to optimize the enzyme concentrations and reaction temperature. Consumption of the more expensive LO enzyme is only 1.4 x 10(-3) U per assay assuming 27 nL per injection. Linearity is established within the range from 0.0025 to 1 mM with R(2) of 0.9982. Recoveries of lactate from five spiked serum samples averaged 101%. Application of this method for the determination of oxalate as an inhibitor of LO is demonstrated.
Qi
Lining
L
Department of Chemistry and Biochemistry, Miami University, Oxford, OH, USA.
Danielson
Neil D
ND
Department of Chemistry and Biochemistry, Miami University, Oxford, OH, USA.
D019075:Electrophoresis, Capillary / Q000379:methods*; D007773:Lactates / Q000032:analysis*; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D010070:Oxalates / Q000737:chemistry*; D009195:Peroxidase / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D007773:Lactates; D010070:Oxalates; D006899:Mixed Function Oxygenases; D009195:Peroxidase; C021497:lactate 2-monooxygenase