Chromatographic separation with subsequent detection of changes in conductivity as a useful tool to detect an increase in the L-dihydroorotic acid (L-DHO) content of cell lysates and serum

A new method for the detection of increased amounts of L-dihydroorotic acid (L-DHO) in Jurkat cell lysates as well as in rat and human serum after chr omatographic separation has been developed which is fast, easily usable, se nsitive and reproducible. The chromatographic separation is per formed usin g an NaOH/water gradient on an anion exchange column. The separated L-DHO p eaks are subsequently measured by detecting the changes of conductivity whe n the amount of L-DHO increases. The validation of the detection method des cribed in that paper showed that in untreated cell and serum samples a peak with the same retention time as L-DHO, was detectable. This probably refle cts the natural content of L-DHO in these samples. No interference was obse rved with of her peaks showing the selectivity of the method developed. L-D HO was stable in Jurkat cells as well as in rat and human serum samples up to 28 days, which was in all cases the longest time period investigated The method proved to be able to quantify the test compound over a range of con centrations of 1.5-150 mu g/ml in the case of cells and 1-30 mu g/ml in the case of serum. The limit of quantification in spiked cell lysates was 1.5 mu g/ml, while in spiked serum if was 1 mu g/ml, with a signal-to-noise rat io of 1.3. In order to investigate the linearity, five calibration curves w ere prepared for each sample and run an five different days showing correla tion coefficients of the regression lines higher than 0.99 and good reprodu cibility of the slope (coefficient of variation [CV] <6.7%). The infer-day accuracy of the measured L-DHO concentration in spiked samples was expresse d as % difference of found to added amount of L-DHO in the different sample s and was found to be within a range of +/-10%. The-inter day precision of the measured L-DHO concentration in spiked Jurkat cells, rat and human seru m, evaluated at three concentrations tin the low, middle and high range of the standard curve) and expressed as CV was better than 6%, 3.7% and 6%, re spectively. This reflects the good reproducibility of the method. Based on these results the new detection method described here can be used for the a nalysis of the in vitro and in vivo effects of compounds that inhibit the a ctivity of dihydroorotate-dehydrogenase (DHODH).