Determination of pyruvate and lactate in primary liver cell culture medium during hypoxia by on-line microdialysis-liquid chromatography.
A microdialysis sampling device was constructed for the measurement of pyruvate and lactate in primary liver cell culture medium during hypoxia. It was composed of a Petri dish, a dialysis membrane and two transmission tubes within a hypoxia chamber. The dialysis membrane was located in the Petri dish such that it was immersed in the culture medium. Dialysates were collected and introduced by an on-line injector to a liquid chromatographic system for analysis of pyruvate and lactate. The detection limit of this assay was 0.2-2.0 microM with acceptable intra- and inter-assay reproducibilities. In order to validate the assay, primary liver cells were incubated in the Petri dish within a hypoxia chamber in an incubator. The baseline concentrations of pyruvate and lactate in primary liver cell culture medium were 10.6+/-5.6 and 607+/-143 microM, respectively. These levels drastically changed during hypoxia and reperfusion. In conclusion, the present assay provides a sensitive, direct measurement of pyruvate and lactate in culture medium while minimizing pretreatment procedures for sample preparation.
Wu
Y S
YS
Department of Environment Engineering, Hung-Kuang Institute of Technology, Taichung, Taiwan.
Tsai
T H
TH
Wu
T F
TF
Cheng
F C
FC
D000818:Animals; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D003470:Culture Media / Q000737:chemistry*; D022781:Hepatocytes / Q000737:chemistry*; D007773:Lactates / Q000032:analysis*; D008297:Male; D017551:Microdialysis; D011773:Pyruvates / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D003470:Culture Media; D007773:Lactates; D011773:Pyruvates