Analytical Approach to Distinguishing Lactate Dehydrogenase Fractions for Oncological Diagnostics

Abstract

Background

One of the crucial enzymes for cancer cell growth is lactate dehydrogenase (LDH, E.C. 1.1.1.27), an oxidoreductase that catalyzes the conversion between pyruvate and lactate. It has been found that in cancer cells metabolism, the LDH isoenzyme profile changes, with forms rich in the muscle-type subunit beginning to dominate over those in which the heart-type predominates. This suggests that by examining changes in the enzymatic activity of isoforms with a specific subunit content, it may be possible to quickly distinguish a physiological sample from a pathological one.

Results

This article focuses on the development of an analytical strategy that enables the estimation of the ratio of LDH fraction activities as a basis for a simple and quick screening test. Spectrophotometric detection of LDH activity is based on the ferrozine photometric reaction with ferrous ions generated during the biocatalytic reduction of ferric ions by NADH. The developed Multicommutated Flow Analysis (MCFA) system, coupled with an optoelectronic flow-through detector, enables the use of a kinetic method based on the inhibition of LDH subunits to monitor the enzyme reaction kinetics. The distinctly different responses of the muscle-type and heart-type subunits to the selected inhibitors revealed a linear relationship between the obtained analytical signal and the percentage content of each subunit. The calibration curves for selected inhibitors are linear within the tested range of standards with coefficients of determination equal to 0.99 each.

Significance

The developed MCFA system was utilized in the analysis of human serum samples obtained from both healthy patients and patients with cancer. The analysis demonstrates that the proposed approach can differentiate oncological serum samples from reference ones based on the LDH fractions activity ratio, even when their total LDH activity level is low.