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 491 nm 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 90 min. The results showed that the new D-lactate assay system had good linearity (R(2)=0.9964) in the calibration range from 5 to 150 μM. 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 90 min, whereas the HPLC method could detect only one sample over the same time period.
Department of Electro-Optical Engineering, National Taipei University of Technology, No. 1, Sec. 3, Chung-Hsiao E. Rd., Taipei 106, Taiwan. Electronic address: cmchen@ntut.edu.tw.
Chien-Ming
CM
Chen
School of Pharmacy, College of Pharmacy, Taipei Medical University, No. 250, Wuxing St., Taipei 11031, Taiwan.
Shih-Ming
SM
Chen
School of Pharmacy, College of Pharmacy, Taipei Medical University, No. 250, Wuxing St., Taipei 11031, Taiwan.
Po-Jen
PJ
Chien
Department of Electro-Optical Engineering, National Taipei University of Technology, No. 1, Sec. 3, Chung-Hsiao E. Rd., Taipei 106, Taiwan.
Han-Yin
HY
Yu
D000818:Animals; D057075:Enzyme Assays / Q000379:methods; D045022:Lactate Dehydrogenases / Q000032:analysis* / Q000378:metabolism*; D019344:Lactic Acid / Q000032:analysis* / Q000378:metabolism*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence / Q000379:methods
D019344:Lactic Acid; D045022:Lactate Dehydrogenases; C071195:D-lactate dehydrogenase
Enzymatic assay system; NADH; UV-light-emitting diode; d-Lactate; d-Lactate dehydrogenase