Determinatioii of enzyme activity in serum by nitrogen laser-excited fluorescence detector

Abstract

Nitrogen laser-excited fluorescence detector (Nisshin Electric Co., Ltd.) is applied to the monitoring of flow injection analysis. The conventional laser-induced technique showed large fluorescence background even with purified water. The major contributions to the blank emission are luminescence from instrumental components, scattered light and Raman scattering from water. But these scattered light are instantaneous and present only while the excitation source irradiates the sample. The combination of pulsed nitrogen laser excitation (peak power : 10 kW) and time resolution in boxcar integrator led to a instrumentation capable of effectively eliminating these contribution to the blank. In relatively pure samples, few problems with this detection system were noted, however, significant interferences were observed in human sera. Fluo- rescence background of serum, which was mainly caused by protein, could be decreased to 20 % of total background by incorporating DEAE-Sepharose column (2 mmX 10 mm) into the flow injection system and by eluting with glycine buffer (pH 9.0) contained 30 mM sodium chloride. Furthermore (1~10) µJ injection of sample which was diluted serum to about seventeen fold with enzymatic agents and buffer solution decreased the residual fluorescence background of serum to negligibly small. In this system, makes it possible to determine NADH which was produced with enzyme reaction in serum. Using this system, concentration of lactic acid and activity acid and activity of lactic dehydrogenase in serum were determined. The reproducibility of the assay using same serum was 13.3mg/dl, standard deviation : 0.65 mg/ dl and coefficient of variation : 4.9% (n=6) for lactic acid and 220 U/L, standard deviation : 12.1 U/L, and coefficient of variation : 5.5 % (n=6) for lactic dehydrogenase, respectively. © 1983, The Japan Society for Analytical Chemistry. All rights reserved.