Determination of pyruvate and lactate in primary liver cell culture mediumduring hypoxia by on-line microdialysis-liquid chromatography

A microdialysis sampling device was constructed for the measurement of pyru vate and lactate in primary liver cell culture medium during hypoxia. it wa s composed of a Petri dish, a dialysis membrane and two transmission tubes within a hypoxia chamber. The dialysis membrane was located in the Petri di sh such that it was immersed in the culture medium. Dialysates were collect ed and introduced by an on-line injector to a liquid chromatographic system for analysis of pyruvate and lactate. The detection limit of this assay wa s 0.2-2.0 muM with acceptable intra- and inter-assay reproducibilities. In order to validate the assay, primary liver cells were incubated in the Petr i dish within a hypoxia chamber in an incubator. The baseline concentration s of pyruvate and lactate in primary liver cell culture medium were 10.6 +/ -5.6 and 607 +/- 143 muM, respectively. These levels drastically changed du ring hypoxia and reperfusion. In conclusion, the present assay provides a s ensitive, direct measurement of pyruvate and lactate in culture medium whil e minimizing pretreatment procedures for sample preparation. (C) 2001 Elsev ier Science B.V. All rights reserved.