ELUCIDATION OF PHASE-I AND PHASE-II METABOLIC PATHWAYS OF RHEIN - SPECIES-DIFFERENCES AND THEIR POTENTIAL RELEVANCE

Because of previously observed species differences in rhein tolerabili ty, with rabbits being very susceptible to kidney disturbances, in viv o and in vitro biotransformation studies were performed to find out wh ether the differences in the undesired effects of rhein are associated with qualitative, species-dependent differences in its metabolism. Fi rst hints on species-dependent biotransformation profiles were obtaine d from in vivo experiments with C-14-labeled rhein in rat, rabbit, dog , and man. TLC-analysis of urine samples obtained after oral administr ation of C-14-rhein to rabbits revealed an additional, hydrophilic met abolite fraction in rabbit urine as compared with dog and human urine, all of which contain phenolic monoglucuronide and monosulfate as majo r metabolites. An investigation of urine samples (obtained from dogs, rabbits, rats, and human volunteers after oral application of unlabele d rhein) was conducted by means of mass spectrometric tandem technique s including on-line HPLC-MS/MS. In vitro experiments with subcellular liver fractions of rats and rabbits revealed the presence of three mon ohydroxylated metabolites of rhein, their quinoid oxidation products, and a bishydroxylated derivative of rhein. The hydroxylated phase I me tabolites were detected as glucuronides in urine samples of all invest igated species, whereas the quinoid product was present only in rabbit urine. Moreover, two regioisomeric phenolic glucuronides and sulfates or glucosides of rhein were found as major phase II metabolites in ur ine of all species. Furthermore, acyl glucuronides of rhein and monohy droxylated rhein and their respective isomeric acyl migration products were identified in human urine. In rabbit urine we discovered differe nt bisglucuronides (bisphenolic glucuronide, mixed ether/ester glucuro nides), whereas in rats only the bisether/ether glucuronide was presen t. In addition, the investigations of dog and human urine showed the f ormation of two regioisomeric phenolic glucosides. With respect to a p otential reactivity with endogenous macromolecules the quinoid metabol ites as well as the bisester/ether glucuronides appear most relevant.