Eva Klasson-Wehler

Competitive inhibition of thyroxin binding to transthyretin by monohydroxy metabolites of 3,4,3',4'-tetrachlorobiphenyl

Abstract In vivo and in vitro inhibition of thyroxin (T 4 )-binding to TTR was observed by monohydroxy-metabolites of 3,4,3′,4′-tetrachlorobiphenyl (TCB). The potency of inhibition followed the order 4-OH-3,5,3′,4′-TCB = 5-OH-3,4,3′,4′-TCB > 2-OH-3,4,3′,4′-TCB > 6-OH-3,4,3′,4′-TCB >>> TCB.

Chromatographic fractionation and analysis by mass spectrometry of conjugated metabolites of bis(2-ethylhexyl)phthalate in urine

Mono(2-ethylhexyl)phthalate (MEHP), the primary metabolite of the plasticizer bis(2-ethylhexyl)phthalate (DEHP), was given to guinea pigs and mice and the methods for the isolation, separation and analysis of its metabolites in urine were developed. Following solid-phase extraction with octadecylsilane-bonded silica, individual metabolites were purified and separated using a combination of ion-exchange chromatography on lipophilic gels and reversed-phase high-performance liquid chromatography. Analysis of intact conjugates, as well as nonconjugated metabolites, was performed by fast atom bombardment mass spectrometry (FAB-MS) and, after derivatization, by gas chromatography-mass spectrometry. Enzymatic methods were used for further characterization. The study confirms glucuronidation as the major conjugation pathway for MEHP in the investigated species. Although less important quantitatively, glucosidation is shown to be an alternative conjugation pathway in mice. The methods developed were applied to a sample of urine from a hyperbilirubinemic newborn infant subjected to DEHP-exposure in conjunction with an exchange transfusion. It was demonstrated that metabolites of DEHP were excreted in amounts which could be analyzed by FAB-MS.

Glucuronidation of mono(2-ethylhexyl)phthalate : some enzyme characteristics and inhibition by bilirubin

A method for assaying mono(2-ethylhexyl)phthalate (MEHP) uridine diphosphate (UDP) glucuronyl transferase activity in microsomal preparations from guinea pig liver is described. The quantitation of the MEHP-glucuronide was performed by HPLC after direct injection of a sample of deproteinized incubation mixture. Solubilization of microsomal UDP-glucuronyltransferase activity was achieved by use of Lubrol, and optimal conditions for glucuronidation of MEHP were established. To investigate whether there is competition between MEHP and bilirubin for glucuronidation, inhibition experiments were performed with solubilized enzyme preparations. In these incubations addition of bilirubin decreased the formation of MEHP-glucuronide. No change in the maximal conversion rate (Vmax) was observed, indicating the occurrence of competitive inhibition. This observation may have implications in clinical situations where patients with hyperbilirubinemia are exposed to MEHP, e.g. in exchange transfusions in newborn infants.

Methylsulfonyl metabolites of xenobiotics can serve as ligands for fatty acid binding proteins in chicken liver and intestinal mucosa

Abstract A fatty acid binding protein (FABP) was isolated from chicken liver and intestinal mucosa which bound [4,4′-bis([ 3 H]methylsulfonyl)-2,2′,5,5′tetrachlorobiphenyl[(CT 3 SO 2 ) 2 TCB] or 3,5-dichlorophenyl [ 14 C]methyl sulfone (DCP[ 14 C]MSO 2 ). These FABPs were isolated and partially purified using gel filtration chromatography. The FABP isolated from chicken intestinal mucosa had an apparent molecular weight of 13,800 Da and was characterized by SDS-polyacrylamide gel electrophoresis and immunoblot analysis to be liver-FABP. The FABP isolated from the liver had an apparent molecular weight of 14,300 Da.