Dawson J, Knowles R G, Pogson C I
Wellcome Research Laboratories, Beckenham, Kent, U.K.
Biochem Pharmacol. 1992 Mar 3;43(5):971-8. doi: 10.1016/0006-2952(92)90601-e.
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.
我们基于将果糖中的(^{14}C)掺入葡糖醛酸共轭物，开发了一种简单且灵敏的方法，用于研究分离的肝细胞中化合物的葡糖醛酸化相对速率。使用禁食大鼠的肝细胞，以尽量减少糖原分解对比活度的任何降低。尽管禁食大鼠分离的肝细胞中葡糖醛酸化速率低于喂食大鼠的肝细胞，这是由于UDP - 葡糖醛酸浓度降低，但仍有可能比较不同化合物的葡糖醛酸化速率。通过在极性氨基氰基柱上进行正相高效液相色谱法，将放射性标记的葡糖醛酸苷与肝细胞产生的([^{14}C])果糖和([^{14}C])葡萄糖分离。发现葡糖醛酸苷的比活度约为([^{14}C])果糖的(50%)。葡糖醛酸苷的绝对量可通过测量肝细胞从果糖产生的([^{14}C])葡萄糖的比活度来确定，该比活度反映了6 - 磷酸葡萄糖的比活度，从而反映了用于葡糖醛酸化的UDP - 葡糖醛酸的比活度，不过对于相对速率的测量来说这并非必要。我们已使用此方法研究分离的大鼠肝细胞中对乙酰氨基酚、4 - 硝基苯酚和1 - 萘酚的葡糖醛酸化动力学。该方法应适用于一系列苷元葡糖醛酸化速率的研究，并且与其他方法不同，不需要葡糖醛酸苷标准品或放射性标记的苷元。
