Recently, we showed that CYP2E1 (cytochrome P450 2E1), an ethanol- inducible cytochrome, plays an important role in caffeine biotransformation, in addition to CYP1A2, especially in the formation of theophylline and theobromine. There was a concurrent discovery of a significant shift of caffeine metabolite profile in the urine of 22 confirmed alcoholics. These observations prompted this proposal to test the feasibility of using the change of caffeine metabolite pattern as a marker of CYP2E1 activity, analogous to the use of a caffeine metabolite ratio as an index for CYP1A2 activity. CYP2E1 has become the target of numerous investigations because of its inducibility by ethanol consumption and its capacity to bioactivate pre- carcinogens (e.g. nitrosamines). Because there is no reference method for the in vivo estimation of CYP2E1 activity, development of methods for in vivo tests must rely on results from in vitro studies. Using pure cytochromes CYP1A2 and CYP2E1 from Vaccinia CDNA expression, we plan to establish that the pattern of specific caffeine metabolites is associated with the ratio of CYP2E1 to CYP1A2 activity. Results obtained with the pure enzymes will then be verified by using human liver microsomes. An index for CYP2E1 activity was proposed to estimate CYP2E1 activity where it yielded a value 10 times higher for 22 alcoholics compared to 27 controls. We proposed to assess this index using results of metabolite profiling of already collected urine samples from 125 confirmed alcoholics and 125 controls. In addition, metabolite patterns and ratios will be scrutinized to search for other CYP2E1 indices. Since alcoholics are known to have highly induced CYP2E1 activity, parameters that show large differences between alcoholics and controls will be evaluated.
