A luminometric assay for determination of ethanol in microdialysates

Abstract

In microdialysis, samples from the extracellular fluid are analysed in the outgoing dialysate. By adding ethanol to the ingoing perfusate and 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 light produced from three coupled enzyme reactions is described. Alcohol dehydrogenase catalyses the oxidation of ethanol with formation of NADH, which is monitored using NADH : FMN oxido­reductase and bacterial luciferase. Each assay can be individually calibrated by measuring the increased rate of NADH formation through the addition of a known amount of ethanol. The linear range of the assay was 0.05 - 10 mmol l^-1 in microdialysate samples. The within-run imprecision was 1.4% CV in 10 mmol l^-1 samples, and ethanol recovery was almost 100%. The assay was applied to microdialysates that were generated from probes implanted in the vastus lateralis muscle in fasting subjects (n = 12) and perfused with 5 mmol l^-1 ethanol at 3 µl min. An outflow/inflow steady-state 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 microcirculatory exchange of solute molecules over the dialysis probe is presented.

• Enzymology Extracellular Space Luminescence Luminometry Microcirculation Microdialysis Perfusion