Analysis of glutathione in endothelial cells grown in 96 well microtitre plates.

Attack of the vascular endothelium by free radical and other reactive oxygen species OCCUTS in many pathological conditions, including rheumatoid arthritis, inflammatory bowel diseases, arteriosclerosis and ischaemd reperfusion injury. Such attack may be an important part of ampllfylng the cellular injury seen. Damage or destruction of endothelial cells will result in activation of thrombotic and inflammatory processes. In addition reactive oxygen species inactivate vital endothelial cell products such as prostacyclin and endothelial derived relaxing factor. Inhibition of these agents alone will lead to thrombotic processes and increased adhesion and activation of neutrophils, with consequential production of more reactive oxygen species. Thus attack of the endothelium may be an important amplification step in mediating reactive oxygen species induced tissue injury. There is thus much interest in studying the role of antioxidants, such as the endogenous antioxidant glutathione (GSH), in protecting endothelial cells against such damage. The enzymatic, colourimetric recycling assay of Tietz is a relatively simple and well-established procedure for measuring GSH levels in a wide variety of cells and tissues [I]. The major disadvantage is the slow throughput of samples dictated by the ability to monitor the rate of colour change in either only a single cuvette at any one time or, at most, six at staggered intervals. We have recently described an automatic procedurc using an auto-analyser that sigruficantly increases sample throughput [2]. We now describe an alternative modified procedure utilising a microtitre plate reader to measure the rate of colour development. Furthermore this method is linked to the measurement of GSH in endothelial cells grown on 96 well microtitre plates. This permits rapid measurement of GSH in endothelial cells grown on such plates with the minimum of procedures and ume. The human endothelial cell line, ECV304 [3], was obtained from the European Collechon of Animal Cell Cultures (ECACC No 92091712). The cells were cultured in MI99 medium, supplemented with 10% heat inactivated fetal calf serum and antibiotics, in 75 cm2 tissue culture flasks at 37OC under an atmosphere containing 5% C02. For the measurement of endothelial cell GSH concentrations, confluent monolayers of endothelial cells were grown on Corning 96 well plates. Cell monolayers were washed twice wlth sem free M199 media. The GSH was released by protein precipitation and cellular disruption achieved by addition of 30pl 1% 5-sulfosalicylic acid to each well. The cell extracts were centrifuged in situ at 1500 rpm, 4OC for 5 min, then 100pl assay buffer ( 1 O O m M sodium phosphate buffer contaimng 5mM sodium EDTA, pH 7.5) added. The sample was either diluted or assayed directly using a loop1 aliquot. This aliquot was transferred to a fresh flat-bottomed 96 well plate containing the standard curve (0-1 nMol GSH/100~1. prepared to give the same final concentrations of 5-sulfosalicylic acid as for the samples). Samples were either assayed neat or after dilution, for example 1 : 1, with assay buffer. To the tests and standards l O O p l of a solution containing 1.5mM 1-l DTNB and 0.5mM 1-1 NADPH in assay buffer were added. The plate reader was blanked on wells containing b d e r and 5-sulfosalicylic acid (250~1) alone. The reaction was inihated by the addition of 50pl 1 U / d glutathione reductase (GRD) to all wells excluding the blank. The reaction was followed at 410nm over 5 minutes using a Dynatech MR5000 plate reader with MR5000/7000 kinetics programme cartridge. Linear rates of reaction were obtained over the range of the standard curve. Quality control samples (QCs) were prepared from confluent cultures of ECV304 endothelial cells after precipitation of protein with 1% 5sulfosalicylic acid. Aliquots of the supernatants were stored in liquid nitrogen. On the day of assay l O O p l assay buffer was added to 30pl aliquot of the QC and l0Opl aliquot assayed. A fresh vial was taken for each assay and each QC was assayed as duplicates taken from the same vial. QCs were assayed in different consecutive assays for inter-assay variauon and in the same assay for intra-assay variation. 0.15 1