110 – Assays for Alcohol and Aldehyde Dehydrogenase: Methods, Approaches, and Applications

This chapter focuses on the methods for the assay of alcohol and aldehyde dehydrogenase. There is widespread research interest in alcohol and aldehyde dehydrogenase enzymes (ADH and ALDH), in an attempt to define their role in alcohol metabolism, and in susceptibility to alcohol-related diseases and as marker for alcoholism. They generate potential carcinogens, and metabolize nonethanol substrates, which may be important in disease pathogenesis, and influence drug efficacy and metabolism. The appropriate method for detecting ADH or ALDH depends on the nature of the question being asked. The researcher may aim to detect as small an amount of enzyme as possible, to mimic a physiological situation in vitro , or perhaps to target the assay at a particular subclass of enzyme. The activity of ADH and ALDH can be assayed using conventional ultraviolet–visible spectrophotometry, but this lacks sensitivity and specificity. Colorimetric techniques—such as use of nitroblue tetrazolium, will improve sensitivity, and N. N-dimethyl-4-nitrosoaniline also improves specificity, as it acts as a co-substrate for ADH. Class-specific fluorogenic substrates have been developed, which have high sensitivity, although the enzyme activities observed with these do not necessarily reflect those of other substrates.