Biochemical Pharmacology, Vol. 41, No. 10, pp. 1493-1496, 1991 0006-2952/91 $3.00 + 0.00 Printed in Great Britain. ~ 1991. Pergamon Press plc GLUCURONIDATION OF MONO (2-ETHYLHEXYL)PHTHALATE SOME ENZYME CHARACTERISTICS AND INHIBITION BY BILIRUBIN PER SJOBERG,*t BORJE EGESTAD,:~ EVA KLASSON-WEHLER§and JAN GUSTAFSSONI[ *Medical Products Agency, Uppsala; ~tDepartment of Physiological Chemistry, Karolinska Institutet, Stockholm, §Environmental Chemistry, Wallenberg Laboratory, University of Stockholm; and IIDepartment of Pediatrics and Pharmaceutical Biochemistry, University of Uppsala, Sweden (Received 28 March 1990; accep~d 15 December 1990) Abstract--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. Exposure to the plasticizer di(2-ethylhexyl)phthalate (DEHP) may occur during a number of situations in medical care where blood products or lipophilic solutions have been in contact with polyvinyl chloride material [1]. Examples of situations where the exposure to DEHP may be of possible significance are exchange transfusions of newborn infants with hyperbilirubinemia, and haemodialysis of patients with renal failure [2, 3]. It is well established that the toxicity of DEHP is not exerted by DEHP itself, but mainly by its mono-deesterified metabolite, mono(2-ethyl- hexyl)phthalate (MEHP) [4]. This compound has been detected in considerable quantities in the blood of patients exposed to DEHP [2, 3]. In fact, it can be formed in vitro during the storage of blood or blood products by the action of non-specific plasma esterases [5]. In experimental animals and in man, elimination of the metabolite MEHP may occur by oxidation or glucuronidation [6]. Since bilirubin is metabolized by glucuronidation, the presence of high circulating blood levels of MEHPin hyperbilirubinemic newborn infants subjected to exchange transfusions [2] raises the question of whether there is competition between MEHP and bilirubin for the same glucuronyltransferase system(s). Such competition may lead to prolonged exposure to MEHP and/or bilirubin. In this paper we describe a method for assaying the formation of MEHP-glucuronide in vitro. Using this method, we have studied some characteristics of the glucuronyltransferase system active on MEHP, including the possible competition t Correspondence should be addressed to: Per Sj6berg, Division of Pharmacology and Toxicology, Medical Products Agency, Box 26, S-751 03 Uppsala, Sweden. between MEHP and bilirubin with regard to glucuronidation. On the basis of observations that guinea pigs eliminate MEHP mainly by glucuronidation [6], solubilized preparations from guinea pig liver microsomes were chosen for the experiments. MATERIALS AND METHODS [14C]Mono(2-ethylhexyl)phthalate ([14C]MEHP) was synthesized from carbonyl-[14C]phthalic anhy- dride (500/~Ci, 58 mCi/nmol, Amersham Interna- tional plc, Amersham, Bucks, U.K.) and 2-ethyl-1- hexanol (0.02nmol, Berol Kemi, Stenungsund, Sweden) by heating the two compounds in 100/zL of toluene at 110° for 6 hr. The reaction mixture was separated by preparative thin layer chromatography (TLC) (SiO2, 60, F254, 0.25 mm with concentration zone, Merck, Darmstadt, F.R.G.) with dichloro- methane/ethyl acetate, 1:1 (v/v) as the mobile phase. Radiochemically pure [14C]MEHP (414/~Ci) was obtained and compared with unlabelled MEHP by TLC. Unlabelled MEHP was synthesized according to a similar procedure [7]. [laC]MEHP-glucuronide was obtained from urine of guinea pigs in which [14C]MEHP had been administered. This species was chosen since it is known to excrete a large fraction of the administered dose as glucuronide [6]. [14C]MEHP was diluted with unlabelled MEHP to give a specific activity of 3.8/zCi/nmol, and two guinea pigs were each given 1.4 nmol/kg body weight of the mixture. Urine was collected on ice for up to 24 hr and the pH of the urine was adjusted to approximately 5. The MEHP-derived metabolites were extracted with octadecylsilane-bonded silica. The extract was 1493