Development of an enzymatic assay system of d-lactate using d-lactate dehydrogenase and a UV-LED fluorescent spectrometer

In this study, we aimed to develop a new enzymatic assay system of d-lactate with good precision, accuracy, and sensitivity for the determination of d-lactate concentrations in rat serum. d-Lactate dehydrogenase (d-LDH) was utilized to catalyze d-lactate and NAD⁺ to pyruvate and NADH, respectively. The generated NADH was excited by using a 340-nm UV-light-emitting diode (LED), and the fluorescence at 491nm was detected to determine the concentration of d-lactate in rat serum. The optics, consisting of the sample cuvette, were set on three-dimensional stages to receive the most intensive fluorescence signal into the spectrometer. The optimal conditions of the d-LDH reaction were pH 8.5 and 25°C for 90min. The results showed that the new d-lactate assay system had good linearity (R²=0.9964) in the calibration range from 5 to 150μM. Intra-day and inter-day accuracies were in the range of 103.96-109.09% and 102.84-104.59%, respectively, and the intra-day and inter-day precision was 4.28-6.82% and 4.04-12.40%, respectively. Finally, serum d-lactate concentrations determined by the proposed enzymatic assay system were compared with those obtained by a conventional HPLC method. The newly developed d-lactate assay system could detect 10-15 samples in 90min, whereas the HPLC method could detect only one sample over the same time period.