Enzymatic Ethanol Methods

Enzymatic measurement of ethanol is the most common method in use in clinical laboratories for quantitation of ethanol. These methods utilize ADH to oxidize ethanol to acetaldehyde with concomitant reduction of NAD+ to NADH, the latter being measured directly at 340 nm.

In addition to measuring the NADH produced in the above reaction, the NADH that is produced may also be utilized in secondary reactions involving colorimetric, fluorometric, or electrochemical detection methods. One such colorimetric method couples the NADH that is produced to the reduction of iodonitrotetrazolium dye by diaphorase. The reduced iodonitrotetrazolium dye can be readily measured at 500 nm.

NADH + iodonitrotetrazolium dye—NAD+ + reduced iodonitrotetrazolium dye (500 nm)

Another detection scheme that has been used is termed radiative energy attenuation (26,27). This fluorometric method is based on the degree of inhibition of fluorescence of fluorescein dye following the production of a colored dye. Thiazolyl blue dye (MTT) reacts with NADH and is reduced resulting in a colored compound with an absorbance at 565 nm. The color of the reduced dye reduces the amount of light interacting with the fluorescein, thereby decreasing its fluorescence. The decrease in fluorescence intensity of the fluorescein dye present in the reagent is inversely related to ethanol present in the sample.

, ADH , NAD+ + ethanol-> NADH + acetaldehyde + H+

Fluorescein , NADH + MMT->NAD+ + reduced MMT

The enzymatic ethanol methods are generally accurate and provide good agreement with chromatographic methods.

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