On-line lactate dehydrogenase substrate and activity determinations by capillary electrophoresis.
Capillary electrophoresis is used to perform both substrate and enzyme assays using the lactate dehydrogenase (LDH) enzyme system. Polymer mediated stabilization with polyethylene glycol (PEG) can extend the lifetime of enzyme activity, increasing the number of assays that can be performed at room temperature. For the determination of lactate, the reduction of the cofactor nicotinamide adenine dinucleotide (NAD+) is monitored at 340 nm, while the determination of pyruvate is accomplished by following the oxidation of the cofactor nicotinamide adenine dinucleotide, reduced form (NADH) at 280 nm. Sample throughput is 1 min, excluding both the wash and equilibrium steps. Linearity is obtained in the clinical range for both lactate and pyruvate standards. In addition, bovine serum with spiked lactate is analyzed with an average recovery of above 90%. For the enzyme assay, the pyruvate reaction is chosen for its more favorable kinetics. Linearity is also established in the clinical range and the examination of serum spiked enzyme samples was possible.
Fujima
J M
JM
Department of Chemistry, Miami University, Oxford, OH 45056, USA.
Danielson
N D
ND
D000818:Animals; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000379:methods; D004795:Enzyme Stability; D007770:L-Lactate Dehydrogenase / Q000032:analysis* / Q000378:metabolism; D007773:Lactates / Q000032:analysis* / Q000097:blood; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D011092:Polyethylene Glycols; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D007773:Lactates; D009243:NAD; D012710:Serum Albumin, Bovine; D011092:Polyethylene Glycols; D007770:L-Lactate Dehydrogenase