Mechanism of formation of 6-amino-5-(N-methylformylamino)-1-methyluracil and 3,7-dimethyluric acid from theobromine in the rat in vitro: involvement of cytochrome P-450 and a cellular thiol.
1. The involvement of glutathione (GSH) and cytochrome P-450 in the conversion of theobromine to 6-amino-5-(N-methylformylamino)-1-methyluracil (3,7-DAU) and 3,7-dimethyluric acid (3,7-DMU) has been investigated in rat liver microsomal incubations. 2. The ratio of formation of 3,7-DAU to 3,7-DMU increased with increasing GSH concentration, reaching a maximum (approximately 12:1) at 2 mM. For any given added GSH concentration the formation of 3,7-DAU plus 3,7-DMU remained constant. 3. 3,7-DAU and 3,7-DMU formation were increased approx. 12- and 1.6-fold in liver microsomes from rats treated with 3-methylcholanthrene and phenobarbitone, respectively. Cimetidine, metyrapone and SKF-525A each inhibited the conversion of theobromine to 3,7-DAU and 3,7-DMU. 4. Apparent Km and Vmax values for the combined formation of 3,7-DAU and 3,7-DMU were the same in the absence and presence of GSH, 2 mM. 5. L-Cysteine and N-acetyl-L-cysteine were as effective as GSH in causing a shift from 3,7-DMU to 3,7-DAU formation, but the non-thiol reducing agents ascorbic acid and alpha-tocopherol were ineffective. 6. Data are consistent with the hypothesis that 3,7-DAU and 3,7-DMU are derived from a common oxidized intermediate of theobromine, the formation of which is rate-limiting. The putative intermediate normally serves as a precursor to 3,7-DMU but in the presence of GSH, or some other cellular thiol, it may be reduced to give 3,7-DAU.
Lelo
A
A
Department of Clinical Pharmacology, Flinders Medical Centre, Bedford Park, Adelaide, Australia.
Birkett
D J
DJ
Miners
J O
JO
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D002927:Cimetidine / Q000494:pharmacology; D003545:Cysteine / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008797:Metyrapone / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013805:Theobromine / Q000378:metabolism*; D014498:Uracil / Q000031:analogs & derivatives* / Q000378:metabolism; D014527:Uric Acid / Q000031:analogs & derivatives* / Q000378:metabolism
D011725:Pyridines; D013438:Sulfhydryl Compounds; C013109:metapyrone; D014527:Uric Acid; C066068:6-amino-5-(N-methylformylamino)-1-methyluracil; D014498:Uracil; D002927:Cimetidine; D003577:Cytochrome P-450 Enzyme System; D005978:Glutathione; D003545:Cysteine; C066065:3,7-dimethyluric acid; D013805:Theobromine; D004229:Dithiothreitol; D000111:Acetylcysteine; D008797:Metyrapone