A LUMINOMETRIC ASSAY FOR DETERMINATION OF ETHANOL IN MICRODIALYSATES

In microdialysis, samples from the extracellular fluid are analysed in the outgoing dialysate. By adding ethanol to the ingoing perfusate an d following its exchange in the dialysate, an outflow/inflow ratio is obtained. This ratio has been shown to correlate with the tissue blood flow. An automatic luminometric ethanol assay that quantifies the lig ht produced from three coupled enzyme reactions is described. Alcohol dehydrogenase catalyses the oxidation of ethanol with formation of NAD H, which is monitored using NADH:FMN oxidoreductase and bacterial luci ferase. Each assay can be individually calibrated by measuring the inc reased rate of NADH formation through the addition of a known amount o f ethanol. The linear range of the assay was 0.05-10 mmol l(-1) in mic rodialysate samples. The within-run imprecision was 1.4% CV in 10 mmol l(-1) samples, and ethanol recovery was almost 100%. The assay was ap plied to microdialysates that were generated from probes implanted in the vastus lateralis muscle in fasting subjects (n=12) and perfused wi th 5 mmol l(-1) ethanol at 3 mu l min(-1). An outflow/inflow steady-st ate ratio of 27% (range 19-47%) appeared after about 1 h, followed by a within-run variability of 4.7% CV (range 2.5-7.7% CV), as calculated from samples collected every 15 min up to 4 h after stabilization. In conclusion, a sensitive ethanol assay, suitable for studies of microc irculatory exchange of solute molecules over the dialysis probe is pre sented.