Photometric flow system for the determination of serum lactate dehydrogenase activity

The routine method for LDH (Lactate dehydrogenase) activity determination is to monitor the increase of NADH concentration at 340 nm. There are some inconvenience in taking measurements in the near-UV region, especially in the case of serum samples analysis. In this work, two modifications of the routine LDH activity assay based on the use of reducing properties of NADH have been compared. Both methods involved the reduction of compounds that can be easily determined by well-known methods, ferric ion (with ferrozine) and nitrotetrazolium blue (NBT). A fully-mechanized Multicommutated Flow Analysis-Paired Emitter Detector Diode (MCFA-PEDD) system based on solenoid devices was developed and applied for both methods. The linear ranges obtained for Fe-ferrozine and NBT methods are 6.0-200.0 U L-1 and 10.0-250.0 U L-1 with estimated detection limits at 0.2 U L-1 and 4.5 U L-1, respectively. The low LOQ values enabled 10-fold sample dilutions, which is advantageous for samples with limited available volume. The Fe-ferrozine method is more selective for LDH activity in the presence of glucose, ascorbic acid, albumin, bilirubin, copper and calcium ions than NBT method. To confirm the analytical usefulness of the proposed flow system, the analysis of real human serum samples was carried out. The statistic tests showed satisfactory correlation between the results obtained for both developed methods and those received using the reference method.

Automated summary

In this study, two modifications of the routine LDH activity assay based on the use of reducing properties of NADH were compared. A fully-mechanized MCFA-PEDD system was developed for accurate measurement of LDH activity in serum samples. The linear ranges obtained for Fe-ferrozine and NBT methods were 6.0-200.0 U L-1 and 10.0-250.0 U L-1 with estimated detection limits at 0.2 U L-1 and 4.5 U L-1, respectively. The Fe-ferrozine method showed higher selectivity for LDH activity in the presence of glucose, ascorbic acid, albumin, bilirubin, copper and calcium ions.