Determination of glucuronidation in isolated rat liver cells by incorporation of 14C from fructose.

Glucuronidation constitutes a major mechanism in the deactivation and elimination of many xenobiotics [ 1, 21. As a general method to study glucuronidation of a range of substrates in intact cells, we have developed an h.p.1.c.-based method for measuring the incorporation of I4C into glucuronides, following the labelling of the UDP-glucuronic acid (UDPGA) pool of isolated liver cells by incubation with 1 l‘Cpructose. Liver cells were prepared [ 3 ] from male Wistar rats starved overnight. Starved rats were used to minimize possible effects of glycogen breakdown on the specific activity of UDPGA. The liver cells were incubated (0.5-2 mg dry weight/ml) with glucuronidation substrates dissolved in dimethylsulphoxide (final concentration 1% v/v) in Krebs buffer containing 200 pwfructose and 0.2-0.5 pCi (‘JClfructose/ml at 37°C for 20-90 min. Incubations were terminated by centrifugation of 1 ml portions through 250 pI silicone oil (Dow Corning S50:dinonyl phthalate, 2 : l ) at 12 000 g for 30 s. Supernatants were heated in a boiling water bath for 5 min before centrifugation. We have modified an h.p.1.c. method that has been used to separate lJC-labelled glucuronides and [ “CIUDPGA [4] for the separation of “C-labelled glucuronides from [ “Cpructose and other radiolabelled reaction products. Supernatants (routinely 0.1 ml) were analysed on a polar amino-cyano column at a flow rate o f 1 ml/min. The mobile phase consisted of a linear gradient from acetonitrile t o 67% 0.0 1 M-tetrabutylammonium hydrogen sulphate (TBAHS) in H,O over 20 min. TBAHS at 67% was maintained for 5 min and the system returned to 100% acetonitrile over the subsequent 10 min. Fractions were collected at 0.5 min intervals for determination of the I4C content. Glucuronide peaks were identified by their disappearance following treatment with B-glucuronidase. The retention time for p-nitrophenol (PNP) and a-naphthol glucuronides was 13 min and for N-acetyl-p-aminophenol (APAP)glucuronide, 15 min. The added [ lJC]fructose and [ “Clglucose, produced in the liver cell incubation, eluted together at 18 min. The sensitivity was 40 pmol per injection, equivalent to 0.4 p~ in the incubations. The amount of glucuronide produced was calculated from the specific activity of the [ 14C]fructose. The amounts of a-naphthol glucuronide formed by liver cellls (100 pM-a-naphthol and 60 min incubations), quantified by the [ lJC]fructose method and by using [ “C]naphthol and the same h.p.1.c. separation were 2.7 nmol/ml and 3.2 nmol/ml, respectively (results from one representative experiment with incubations in duplicate).