CONTINUOUS FLUOROMETRIC ASSAY FOR HUMAN ALDEHYDE DEHYDROGENASE AND ITS APPLICATION TO BLOOD ANALYSIS

A highly fluorogenic synthetic substrate for human ''low K-m'' aldehyd e dehydrogenase (ALDH) isozymes, 7-methoxy-1-naphthaldehyde, was ident ified. The new substrate is characterized by submicromolar or low micr omolar Michaelis constants (K-m) for both cytosolic and mitochondrial ALDH from the human liver. The cytosolic ALDH oxidizes the naphthaldeh yde substrate with a rate almost equal to that of the respective aceta ldehyde reaction, while the mitochondrial isozyme is ca. 25 fold less active. Thanks to a high fluorescence yield of the oxidation product, i.e., the corresponding naphthoate (phi = 0.41), reaction rates as low as 0.1 nM min(-1) can be measured reproducibly. The emission spectral characteristics of the naphthoate product differ significantly from t hat of the aldehyde (lambda(max) at 395 and 475 nm, respectively), all owing a selective observation of the former, and there is also marked difference between the naphthoate and the corresponding alcohol (lambd a(max) at 355 nm), helping to eliminate possible interferences from al cohol dehydrogenase and/or aldehyde reductase in the fluorimetric assa y. As a possible application of the new assay, it is shown that the AL DH activity can easily be detected, by continuous monitoring of the fl uorescence increase, in 1000-fold diluted hemolysates, using 7-methoxy -1-naphthaldehyde and NAD(+) as substrates, and there is no necessity for prior removal of hemoglobin. Normal ALDH level in the blood of hea lthy volunteers has been measured, and the result, 4.9 U l(-1) (n = 27 ), agrees with the literature data obtained for the acetaldehyde oxida tion activity, and exceeds by more than 20-fold the lower detection li mit of the method. The present method is free of a background drift, c haracteristic of all the previously proposed spectral assays for ALDH.