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Identification of lipid peroxidation products as physiological substrates of aldo-keto

4342 Aldo-keto reductase 1 B10 (AKR1B10, also designated aldose reductase-like-1, ARL-1), overexpressed in human liver, lung, and breast cancers, possesses active-site properties of a carbohydrate binding protein. Recent studies demonstrate that AKR1B10 facilitates cancer cell proliferation and clonogenic growth. The current study determines the substrate specificity of AKR1B10 and validates lipid peroxidation products as its efficient physiological substrates. In this observation, recombinant AKR1B10 protein was prepared using a prokaryotic protein expression system and used for enzymatic activity assays. Kinetic constants (K m and V max ) were determined using colorimetric methods with substrates at a range of 1-20,000 µM. Results show 4-hydroxynonenal (HNE), crotonaldehyde, acrolein, trans-2-hydroxenal, and 2,4-hexadienal are all ideal substrates of AKR1B10, with K m at 24.0 to 110.0 µM and V max at 1,559.0 to 5,837.0 nmol/mg protein/min. To estimate physiological significance, the lowest active concentrations of these substrates were defined by high-performance liquid chromatography (HPLC) analysis of the enzymatic products; and results showed that efficient reductions occurred at 900 nM for crotonaldehyde, 800 nM for acrolein, 200 nM for HNE, 200 nM for trans-2-hydroxenal, and 100 nM for 2,4-hexadienal, all of which are at or close to the pathophysiological concentrations to which humans expose. These data suggest that AKR1B10 is a critical enzyme that eliminates highly toxic reactive carbonyls produced during cell metabolism, being a potential target for cancer management.