FLUOROMETRIC DETECTION OF ALDEHYDE DEHYDROGENASE-ACTIVITY IN HUMAN BLOOD, SALIVA, AND ORGAN BIOPSIES AND KINETIC DIFFERENTIATION BETWEEN CLASS-I AND CLASS-III ISOZYMES

Two highly fluorogenic aldehydes, 7-methoxy-1-naphthaldehyde (MONAL-71 ) and 6-methoxy-2-naphthaldehyde (MONAL-62), were examined as indicato rs of the aldehyde dehydrogenase (ALDH) activity in human tissue homog enates and accessible body fluids. Both compounds were previously foun d to be excellent substrates for the ALDH from erythrocytes and for th e purified class I(cytosolic) ALDH from human Liver. By contrast, only MONAL-62, but not the isomeric MONAL-71, was oxidized by class IH ALD H present in human saliva. The apparent K-m for the former compound re acting with saliva ALDH is 0.24 mu M, with the reaction rate (V-max) c lose to that of benzaldehyde oxidation. There is also a fully competit ive inhibition of the fluorogenic oxidation of the MONAL-62 by benzald ehyde. Both NAD(+) and NADP(+) can be used as oxidants in this reactio n, with comparable rates, a fact previously reported for the human cla ss III aldehyde dehydrogenase. In human liver homogenate (cytosolic microsomal fraction), the ALDH activity is easily detectable using eit her MONAL-71 or MONAL-62, with specific activities of approximately 2. 5 and 3.2 units per gram of protein, respectively. The low apparent K- m values, 0.85 and <0.03 mu M, respectively, together with the inhibit ion profile by propionic aldehyde (ID50 in the micromolar range) indic ate that both compounds are oxidized primarily by the class I ALDH, fu rther confirmed by low activity (0.4 U/g) with NADP(+) as oxidant. By contrast, in human stomach, containing mostly class III ALDH, the acti vity measured with MONAL-71, 0.4 U/g, is much lower than that with MON AL-62 (5.1 U/g with NAD(+) and 3.1 U/g with NADP(+)), the latter being virtually insensitive to 1 mM propionic aldehyde. Hence, in a stomach homogenate, class I and class III ALDH activities can be measured sel ectively with the two fluorogenic substrates described. In all experim ents, the activity of aldehyde oxidase was at least 10-fold lower than that of the ALDH. Addition of 5 mM 4-methylpyrazole, a known inhibito r of the alcohol dehydrogenase, did not change the resultant ALDH acti vities by more than 10%, indicating lack of interference by the former enzyme. A preliminary screening of two liver tumour samples showed di minished class I ALDH activities (0.7 and 0.03 U/g), but no evidence f or class III ALDH induction. The above observations are discussed in r elation to the mechanism of detoxication of cyclophosphamide. (C) 1997 Academic Press.