Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 150-155 |
| Number of pages | 6 |
| Journal | Journal of Pharmaceutical and Biomedical Analysis |
| Volume | 116 |
| DOIs | |
| Publication status | Published - Mar 2 2015 |
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Keywords
- D-Lactate
- D-Lactate dehydrogenase
- Enzymatic assay system
- NADH
- UV-light-emitting diode
ASJC Scopus subject areas
- Analytical Chemistry
- Drug Discovery
- Pharmaceutical Science
- Spectroscopy
- Clinical Biochemistry
Cite this
Development of an enzymatic assay system of d-lactate using d-lactate dehydrogenase and a UV-LED fluorescent spectrometer. / Chen, Chien Ming; Chen, Shih Ming; Chien, Po Jen; Yu, Han Yin.
In: Journal of Pharmaceutical and Biomedical Analysis, Vol. 116, 02.03.2015, p. 150-155.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Development of an enzymatic assay system of d-lactate using d-lactate dehydrogenase and a UV-LED fluorescent spectrometer
AU - Chen, Chien Ming
AU - Chen, Shih Ming
AU - Chien, Po Jen
AU - Yu, Han Yin
PY - 2015/3/2
Y1 - 2015/3/2
N2 - 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.
AB - 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.
KW - D-Lactate
KW - D-Lactate dehydrogenase
KW - Enzymatic assay system
KW - NADH
KW - UV-light-emitting diode
UR - http://www.scopus.com/inward/record.url?scp=84945457107&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84945457107&partnerID=8YFLogxK
U2 - 10.1016/j.jpba.2015.07.018
DO - 10.1016/j.jpba.2015.07.018
M3 - Article
VL - 116
SP - 150
EP - 155
JO - Journal of Pharmaceutical and Biomedical Analysis
JF - Journal of Pharmaceutical and Biomedical Analysis
SN - 0731-7085
ER -