TABLE 1
Enantiomer composition of NNAL produced from NNK by oxidoreductases purified from human liver fractions: comparative analysis by chiral HPLC and by separation of diastereomeric MTPA-NNAL
NNK was incubated in a volume of 1 or 2 ml with the enzyme and a NADPH-generating system for 60 min at 37°C. NNAL was extracted with ethyl acetate, purified by TLC, and divided for enantiomer analysis by the two procedures as detailed under Materials and Methods. NNK conversion to NNAL by cytosolic oxidoreductases amounted to 8 to 12% except for AKR1C4 with 36%. NNAL produced by 11β-HSD1 corresponded to 0.8% of the substrate and was purified by gradient HPLC. At least three additional analyses using enzyme preparations from different liver samples were done with AKR1C1, AKR1C2, and CR I. The (R)-NNAL values found did not deviate by more than 1.5% (in absolute terms) from those displayed.