Materials and Methods

Methods are included in the text and figure legends. β-d-glucuronidase (Part G-7396), α-napthyl β-d-glucuronide, β-trifluoromethylumbelliferyl β-d-glucuronide, β-estradiol 3-(β-d-glucuronide), p-acetominophenyl β-d-glucuronide, 5β-androstane-3α,17α-diol-11-one-17-carboxilic acid-3-(β-d-glucuronide), uridine 5′-diphosphoglucuronic acid (UDPGA), and 4-methylumbelliferone β-d-glucuronide were obtained from Sigma Chemical Co. (St. Louis, MO). Tetrahydrocortisone 3-β-d-glucuronide was obtained from Molecular Probes (Eugene, OR). Recombinant control and UGT1A6 membrane preparations were generated at PanVera Corporation and are commercially available from PanVera (Madison, WI). All other reagents were analytical grade or better and purchased from a variety of commercial sources.

Results and Discussion

We sought to develop an assay that can be used to demonstrate the presence of β-d-glucuronides that might be generated from different classes of UGT enzymes. An assay based on β-glucuronidase activity provides a high throughput screening method to identify structurally different β-d-glucuronides. Cleavage of 4-methylumbelliferyl β-d-glucuronide yielded the highly fluorescent compound 4-methylumbelliferone. Under linear conditions of protein concentration and incubation time, the apparentK_m value for cleavage of 4-methylumbelliferyl-β-d-glucuronide was approximately 56 μM (Fig. 1).

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Figure 1

Kinetic measurements of 4-methylumbelliferylβ-d-glucuronide cleavage by β-glucuronidase.

A, 100-μl assays were performed in triplicate using 100 mM Tris-HCl buffer, pH 7.4, containing 10 mM MgCl₂; 0.1 μg/ml β-glucuronidase; and 5 (▪), 20 (▴), and 100 (●) μM, 4-methylumbelliferyl-β-d-glucuronide. Reactions were started by addition of enzyme, incubated for indicated times at ambient temperature, and then quenched with 100 μl of 0.1 M Na₂CO₃, pH 9.5, containing 0.1% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate. Fluorescence intensity was measured with a Beacon fluorescent polarization instrument (PanVera Corp.) in intensity mode, using excitation and emission filters of 360 nm and 440 nm, respectively, and slit widths set at 10 nm. B, assays were performed in duplicate for 20 min as above with indicated amount of 4-methylumbelliferyl-β-d-glucuronide. 4-Methyumbelliferone concentration was determined from a calibration curve.

To show the feasibility of developing a high throughput inhibition assay, we examined the potential of a variety of structurally dissimilar commercially available β-d-glucuronides to act as inhibitors for the cleavage of 4-methylumbelliferyl-β-d-glucuronide. These β-d-glucuronides represent compounds that might potentially be formed by UGT activity, e.g., phenol and steroid glucuronides. Figure 2 shows that all of the β-glucuronides tested inhibited the production of 4-methylumbelliferone by β-glucuronidase. The potential to inhibit 4-methylumbelliferyl-β-d-glucuronide cleavage appeared to be dependent on the chemical nature of the substituted aglycone, with β-estradiol-3-(β-d-glucuronide) and α-naphthyl β-d-glucuronide showing the strongest and weakest inhibition, respectively. UDPGA, an essential cofactor for UGTs, was not an effective competitor over the range tested, probably due to the α configuration of the sugar bond to UDP (Parkinson, 1996).

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Figure 2

Inhibition curves.

Assays were performed as described in the legend to Fig. 1B with 20 μM 4-methylumbelliferyl-β-d-glucuronide and indicated amount of competitor β-d-glucuronide. Fluorescence intensity due to 4-methylumbelliferyl-β-d-glucuronide cleavage, in the absence of a competitor, was arbitrarily assigned as 100%. Competitors, α-napthyl β-d-glucuronide (▪), β-trifluoromethylumbelliferyl -β-d-glucuronide ([ciro]), β-estradiol 3-(β-d-glucuronide) (▴),p-acetominophenyl β-d-glucuronide (●), 5β-androstane-3α,17α-diol-11-one-17-carboxylic acid-3-(β-d-glucuronide) ([itrf]), and UDPGA (■) were dissolved in 100 mM Tris-HCl, containing 10 mM MgCl₂, pH 7.5. Tetrahydrocortisone 3-β-d-glucuronide (♦) was prepared in dimethyl sulfoxide. Dimethyl sulfoxide in reaction mixtures was 0.1% or less.

To evaluate whether the fluorescent assay could be used to detect β-glucuronides generated from a biochemical assay, two approaches were taken. First, α-naphthyl β-d-glucuronide was generated in an incubation with recombinant human UGT1A6 and α-naphthol. Following extraction, 4-methylumbelliferyl-β-d-glucuronide assay reagents were added directly to the extracted residues. In a separate experiment, it was shown that aglycones are extracted with ethyl acetate and hence transferred to the β-glucuronidase reporter assay. Figure 3A shows that residues from the UGT1A6 assay but not the control microsome assay inhibited 4-methylumbelliferyl-β-d-glucuronide cleavage. Residues from the zero time point extractions do not appear to inhibit the 4-methylumbelliferone cleavage assay, which indicated that the aglycone α-naphthol does not interfere nonspecifically with this assay under these conditions. Inhibition of 4-methylumbelliferyl-β-d-glucuronide cleavage was dependent on the incubation time of the UGT1A6 assay, presumably reflecting increased α-naphthyl glucuronide accumulation. (Accumulation of α-naphthyl glucuronide in the UGT1A6 assay was independently verified in a parallel experiment using [¹⁴C]UDPGA; data not shown.) In the second approach, 4-methylumbelliferyl-β-d-glucuronide assay reagents were added directly to an α-naphthyl glucuronidation assay that was performed in a 96-well plate. Figure 3B shows that 4-methylumbelliferyl β-d-glucuronide cleavage was 60% slower when added to the UGT1A6 assay compared with the control. The homogeneous format circumvented the need for extraction but required longer incubation periods with the recombinant enzyme. These results demonstrate the following: 1) The 4-methylumbelliferyl-β-d-glucuronide assay can be used to detect β-d-glucuronides generated in biological preparations; 2) The choice of α-naphthol β-d-glucuronide, which is a weak competitive inhibitor (Fig. 2), adds credence to this assay. More potent inhibitors such as estradiol β-d-glucuronide (Fig. 2) are likely to be more effective; 3) The assay is amenable to a high throughput format as a two-step procedure; and 4) Heterologous expression systems are useful for the generation of high concentrations of β-d-glucuronides, such as α-naphthyl glucuronide, which was a weak competitor (Fig. 2), in the β-d-glucuronide cleavage assay.

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Figure 3

Inhibition of 4-methylumbelliferyl-β-d-glucuronide cleavage by α-naphthyl glucuronide produced by recombinant UGT1A6.

Both experiments were carried out as a two-step procedure. First an incubation was performed to generate α-naphthol glucuronide, then the fluorescent cleavage assay was performed. Incubation times reflect only glucuronide generation and not time for performing the fluorescent cleavage assay. A, glucuronidation assays with 0.6 mg/ml recombinant UGT1A6 (solid columns) and control (cells infected with a wild-type baculovirus; empty columns) insect cell extracts, were performed for the indicated times in 100 mM Tris-HCl, pH 7.4, containing 10 mM MgCl₂, 2 mM UDPGA, 0.6 mM α-naphthol, at 37°C. Aliquots (0.15 ml) were removed and extracted with 1.5 ml of ethyl actetate and dried down to a residue. 4-Methylumbelliferyl-β-d-glucuronide assay reagents were added directly to the residues and the reaction was performed as described in Fig. 1B for 30 min. Fluorescence intensity due to 4-methylumbelliferyl-β-d-glucuronide cleavage with a residue obtained from an UGT1A6 incubation in the absence of α-naphthol was arbitrarily assigned as 100%. Relative fluorescence intensity of 4-methylumbelliferyl-β-d-glucuronide cleavage assays with residues from glucuronidation incubations, which contained α-naphthol, is shown. B, glucuronidation assays were performed in triplicate in a 96-well plate, as in A, for indicated times, in a total volume of 80 μl. Ten microliters each of 100 μM 4-methylumbelliferyl-β-d-glucuronide and 1 μg/ml β-glucuronidase was then added to each assay. 4-Methylumbelliferyl-β-d-glucuronide cleavage was measured as described in the legend to Fig. 1B using a PolarStar 96-well plate analyzer (BMG, Offenburg, Germany) with slit widths at 20 nm and excitation and emission wavelengths of 360 and 450 nm, respectively. Fluorescence from an incubation with UGT1A6 in the absence of α-naphthol was arbitrarily assigned as 100%. Relative fluorescence intensity of assays from UGT1A6 (solid columns) and control (empty columns) incubations, which include α-naphthol, is shown.

Some caveats to performing this type of assay should be mentioned. The rate of cleavage is important and compared with O-linked glucuronides, N-linked glucuronides appear to be cleaved more slowly by β-d-glucuronidase (Parkinson, 1996). Also, carboxylic acid β-d-glucuronides form reactive acyl-glucuronides that could form covalent linkages to assay components (Parkinson, 1996). The use of human biological tissue preparations to generate β-glucuronides might be complicated by several factors including the presence of naturally occurring fluorescent compounds and the common use of saccharolactone to inhibit levels of endogenous β-glucuronidase. The type of β-glucuronidase and the reaction conditions (such as pH) are important (Hawes, 1998). Cleavage by β-glucuronidase may result in compounds with fluorescent properties similar to that of 4-methylumbelliferone. This problem could be solved by either adding an aglycone control or performing a second β-glucuronidase assay, in parallel with the 4-methylumbelliferyl β-d-glucuronide assay, which used a substrate that had different fluorescent emission properties, e.g., resorufin β-d-glucuronide. In addition, organic extraction was used to obtain glucuronides from the UGT assay in the experiment described in Fig. 3A. Extraction can be avoided, as shown in Fig 3B. However, using a solid-phase extraction method in a 96-well format or centrifugation to remove microsomal membranes would also be more consistent for high throughput assays. Also, replacing 4-methylumbelliferone β-d-glucuronide with a lower affinity substrate in the β-glucuronidase assay may improve the potential of the assay for a high throughput format.

In summary, we demonstrated the feasibility of using a fluorescent assay that can be adapted to a high throughput format to measure the presence of β-d-glucuronides. The assay described here uses relatively inexpensive reagents and does not require chromatographic resolution (thin-layer chromatography or HPLC) or radioactivity. The assay can be performed in a homogeneous format and could be easily automated. The assay will not eliminate the usefulness of other analytical procedures needed to determine the kinetic parameters of glucuronidation, but shows potential as a rapid preliminary screening method similar to that developed for cytochrome P-450 (Crespi et al., 1997). In addition, the principles and procedures described here could be applied to other important classes of phase II conjugating enzymes. For example, sulfate conjugates from sulfonation assays would be expected to compete in a similarly designed assay that used a sulfatase to generate a fluorescent product.