Measurement of glucuronidation by isolated rat liver cells using [14C]fructose.
We have developed a simple and sensitive method for the study of the relative rates of glucuronidation of compounds, in isolated liver cells, based on the incorporation of 14C from fructose into glucuronide conjugates. Liver cells from fasted rats are used to minimize any reduction of the specific activity by glycogenolysis. Although rates of glucuronidation are lower in isolated liver cells from fasted rats than in those from fed rats, because of a reduction in the concentration of UDP-glucuronic acid, it is possible to compare the rates of glucuronidation of different compounds. Radiolabelled glucuronides are separated from [14C]fructose and [14C]glucose, produced by the liver cells, by normal-phase HPLC on a polar amino-cyano column. The specific activity of the glucuronide was found to be approximately 50% of that of the [14C]fructose. Absolute amounts of glucuronide can be determined by measuring the specific activity of the [14C]glucose, also produced by liver cells from fructose, which reflects that of the glucose-6-phosphate and hence the UDP-glucuronic acid used for glucuronidation, although for the measurement of relative rates this would not be necessary. We have used this method to examine the kinetics of the glucuronidation of N-acetyl-p-aminophenol (acetaminophen), 4-nitrophenol and 1-naphthol in isolated rat liver cells. The method should be applicable to the study of the rates of glucuronidation of a range of aglycones and, unlike other methods, does not require glucuronide standards or radiolabelled aglycone.
Dawson
J
J
Wellcome Research Laboratories, Beckenham, Kent, U.K.
Knowles
R G
RG
Pogson
C I
CI
D000082:Acetaminophen / Q000378:metabolism; D000818:Animals; D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005632:Fructose / Q000494:pharmacology*; D005964:Glucosyltransferases / Q000378:metabolism; D005965:Glucuronates / Q000032:analysis; D014453:Glucuronosyltransferase / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D009284:Naphthols / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D002250:Carbon Radioisotopes; D005965:Glucuronates; D009284:Naphthols; D009596:Nitrophenols; C029350:1-naphthol; D005632:Fructose; D000082:Acetaminophen; D005964:Glucosyltransferases; D014453:Glucuronosyltransferase; C059055:N-acetylglucosaminyldiphosphoundecaprenol glucosyltransferase; C024836:4-nitrophenol