Microsomal quercetin glucuronidation in rat small intestine depends on age and segment

Abstract

UDP-glucuronosyltransferase (UGT) activity toward the flavonoid quercetin and UGT protein were characterized in three equidistant small intestine (SI) segments from 4-, 12-, 18-, and 28-month-old male Fischer 344 rats (n = 8/age) using villin to control for enterocyte content. SI microsomal intrinsic clearance of quercetin was increased 3- to 9-fold from 4 months in the proximal and distal SI at 12 and 18 months. Likewise, at 30 μM quercetin, SI microsomal glucuronidation activity was increased with age: 4.8- and 3.9-fold greater at 18 months than at 4 months. Quercetin UGT regioselectivity was not changed by age. The distal SI preferentially catalyzed glucuronidation at the 7-position, whereas the proximal SI produced the greatest proportion of 4'- and 3'-conjugates. Enterocyte UGT content in different SI segments was not consistently changed with age. In the proximal SI, UGT1A increased 64 and 150% at 12 and 18 months and UGT1A1, UGT1A7, and UGT1A8 were also increased at 12 and 18 months. However, age-related changes in expression were inconsistent in the medial and distal segments. Microsomal rates of quercetin glucuronidation and UGT expression were positively correlated with UGT1A1 content for all pooled samples (r = 0.467) and at each age (r = 0.538-0.598). UGT1A7 was positively correlated with total, 7-O- and 3-O-quercetin glucuronidation at 18 months. Thus, age-related differences in UGT quercetin glucuronidation depend on intestinal segment, are more pronounced in the proximal and distal segments and may be partially related to UGT1A1 and UGT1A7 content.

Fig. 1.

Relative cytosolic villin content of tissue preparations at different ages and rat SI segment. Data are means ± S.E.M.; n = 8/age. Log-transformed data were analyzed by two-way ANOVA. P < 0.0003 for section and age.

Fig. 2.

Age-related changes in villin-adjusted kinetics of quercetin glucuronidation, as the sum of all metabolites, by pooled microsomal protein fractions from proximal (A), medial (B), and distal (C) SI segments of male F344 rats. Solid lines indicate that a Michaelis-Menten model and dashed lines indicate that an uncompetitive inhibition model were best fits for data as specified under Materials and Methods.

Fig. 3.

Villin-adjusted rates of microsomal glucuronidation of 30 μM quercetin by SI segment and age with 0.2 mg of microsomal protein/ml and a 30-min incubation. Data are means of the sum of metabolites ± S.E.M.; n = 8/age. Log-transformed data were analyzed by two-way ANOVA. P < 0.0001 for age and segment, and P = 0.3050 for interaction between age and segment.

Fig. 4.

Villin-adjusted rates of glucuronidation of 30 μM quercetin by microsomal fractions collected from mucosa of SI segment with 0.2 mg of microsomal protein/ml and a 30-min incubation. A, proximal; B, medial; C, distal. Data are means ± S.E.M. of n = 8/age. Log-transformed data were analyzed by two-way ANOVA. gluc, glucuronide.

Fig. 5.

Regioselectivity at 30 μM quercetin in small intestine is independent of age. Data are means ± S.E.M.; n = 32. gluc, glucuronide. ^a–c, Means without sharing the same letter in the same metabolite group are different.

Fig. 6.

Standard dilution curves and representative Western blots. A, UGT1A using recombinant human UGT1A1 for a standard curve and 37.5 μg/well of rat proximal SI microsomal protein at different ages. B, UGT1A1 using mixtures of rat SI microsomal samples with high and low responses at 37.5 μg/well and 37.5 μg/well distal SI microsomes from 4- and 28-month-old rats. C, UGT1A7 using dilutions of rat SI microsomal samples with a high response and 37.5 μg/well of proximal SI microsomal protein from 4- and 28-month-old rats. D, UGT1A8 using dilutions of a rat SI microsomal sample with a high response and 37.5 μg/well of distal SI microsomal protein from 4- and 28-month-old rats.

Fig. 7.

UGT1A (A), UGT1A1 (B), UGT1A7 (C), and UGT1A8 (D) enterocyte protein content [(UGT density)/(villin density)] assessed by Western blotting of equidistant small intestine mucosa from 4-, 12-, 18-, and 28-month-old rats. Data were analyzed by two-way ANOVA; n = 8/age. Log-transformed values were used. P values for UGT1A were 0.3064 for age, <0.0001 for segment, and 0.0012 for their interaction, for UGT1A1 were 0.0763 for age, <0.0001 for segment, and 0.0799 for their interaction, for UGT1A7 were <0.0001 for age, <0.0001 for segment, and 0.2668 for their interaction, and for UGT1A8 were 0.9585 for age, 0.0008 for segment, and 0.0034 for their interaction. Representative Western blots and semiquantitative standard curves for each antibody are presented in Fig. 6.