Effect of the β-glucuronidase inhibitor saccharolactone on glucuronidation by human tissue microsomes and recombinant UDP-glucuronosyltransferases

Abstract

Glucuronidation studies using microsomes and recombinant UDP-glucuronosyltransferases (rUGTs) can be complicated by the presence of endogenous β-glucuronidases leading to underestimation of glucuronide formation rates. Saccharolactone is the most frequently used β-glucuronidase inhibitor, although as of yet it is not clear whether this reagent should be routinely added to glucuronidation incubations. Here we determined the effect of saccharolactone on eight different UGT probe activities using pooled human liver microsomes (pHLMs) and rUGTs. Despite the use of buffered incubation solutions it was necessary to adjust the pH of saccharolactone solutions to avoid effects (enhancement or inhibition) of lowered pH on UGT activity. Saccharolactone at concentrations ranging from 1 to 20 mM failed to show enhancement of any of the glucuronidation activities evaluated that could be considered consistent with inhibition of β-glucuronidase. However, for most activities, higher saccharolactone concentrations resulted in a modest degree of inhibition. The greatest inhibitory effect was observed for 5-hydroxytryptamine and estradiol glucuronidation by pHLMs with 35% decrease at 20 mM saccharolactone concentration. Endogenous β-glucuronidase activities were also measured using various human tissue microsomes and rUGTs with estradiol-3-glucuronide and estradiol-17-glucuronide as substrates. Glucuronide hydrolysis was observed for pHLMs, lung microsomes, and insect-cell expressed rUGTs, but not for kidney or intestinal microsomes, or HEK293 microsomes. However, the extent of hydrolysis was relatively small representing only 9 to 19% of the glucuronide formation rate measured in the same preparations. Consequently, these data do not support the routine inclusion of saccharolactone in glucuronidation incubations and, if used, saccharolactone concentrations should be titrated to achieve activity enhancement without inhibition.

Characterization of Inhibitory Effect of Carbapenem Antibiotics on the Deconjugation of Valproic Acid Glucuronide

“…Nakamura et al (2008) used water as an incubation medium for the inhibition study with SL, whereas we used buffered solution (pH 7.4) throughout the experiments. Because SL is an acidic compound, the pH of the incubation medium may be decreased by the addition of 10 mM SL to water, and this may result in nonspecific inhibition (Oleson and Court, 2008). In accordance with this hypothesis, we found complete inhibition of VPA-G deconjugation activity by 10 mM SL in water and confirmed that the pH of the incubation medium was as low as pH 2 under this condition.…”

“…After a 15-min preincubation of a reaction mixture (72 l) consisting of 1.6 mg/ml human or rat liver cytosol and inhibitors (50 M MEPM or 2 mM SL in 100 mM potassium phosphate buffer, pH 7.4, at 37°C), enzyme reactions were initiated by adding 8 l of substrates: final 50 M VPA-G, 100 M 4-MUG, or 200 M MFA-G. Concentrations of MEPM and SL were selected to completely inhibit the deconjugation reaction of MEPM (see Results) and ␤-glucuronidase (Oleson and Court, 2008), respectively. After incubation at 37°C in a shaking water bath for 40 min, the reaction was terminated by transferring the 20 l aliquot to another tube containing 70 l of water, 10 l of HClO 4 , and 10 l of hexanoic acid (100 M) (internal standard) and then 10 l of KHCO 3 (2 M) was added for neutralization.…”

Phase II Metabolism in Human Skin: Skin Explants Show Full Coverage for Glucuronidation, Sulfation,N-Acetylation, Catechol Methylation, and Glutathione Conjugation

“…Addition of 1 mM UDPGA, PAPS, AcCoA, and SAM did not significantly enhance the reaction rates of glucuronidation, sulfation, N-acetylation, and catechol methylation, respectively. Similarly, the inclusion of 1 mM D-saccharolactone, an inhibitor of b-glucuronidase (Oleson and Court, 2008), did not affect the glucuronidation rate of 4-methylumbelliferone. Since conjugation with glutathione may also proceed as a nonenzymatic reaction (Satoh, 1995), the assays were not performed in a medium supplemented with glutathione.…”

Reaction Phenotyping: Advances in the Experimental Strategies Used to Characterize the Contribution of Drug-Metabolizing Enzymes

“…In some instances saccharolactone has been shown to preserve the glucuronide (Bauman et al, 2005). However, in other instances, saccharolactone did not increase glucuronide formation rates, and in fact, even lowered the glucuronide formation levels (Kaivosaari et al, 2008;Oleson and Court, 2008;Walsky et al, 2012a). It is the authors' opinion and experience that saccharolactone is not necessary due to its potential detrimental effects; hence, it can be excluded from in vitro incubations.…”