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In Vitro Glucuronidation of Thyroxine and Triiodothyronine by Liver Microsomes and Recombinant Human UDP-Glucuronosyltransferases

Biotransformation Division (Z.T., H.L., A.C.) and Discovery Analytical Chemistry (I.G., O.M.), Wyeth Research, Collegeville, Pennsylvania

(Received May 31, 2007; accepted September 12, 2007)

	Abstract

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Glucuronidation, which may take place on the phenolic hydroxyl and carboxyl groups, is a major pathway of metabolism for thyroxine (T4) and triiodothyronine (T3). In this study, a liquid chromatography/mass spectrometry (LC/MS) method was developed to separate phenolic and acyl glucuronides of T4 and T3. The method was used to collect the phenolic glucuronide of T4 for definitive characterization by NMR and to determine effects of incubation pH, species differences, and human UDP-glucuronosyltransferases (UGTs) involved in the formation of the glucuronides. Formation of T4 phenolic glucuronide was favored at pH 7.4, whereas formation of T4 acyl glucuronide was favored at pH 6.8. All the UGTs examined catalyzed the formation of T4 phenolic glucuronide except UGT1A4; the highest activity was detected with UGT1A3, UGT1A8, and UGT1A10, followed by UGT1A1 and UGT2B4. Formation of T3 phenolic glucuronide was observed in the order of UGT1A8 > UGT1A10 > UGT1A3 > UGT1A1; trace activity was observed with UGT1A6 and UGT1A9. UGT1A3 was the major isoform catalyzing the formation of T4 and T3 acyl glucuronides. In liver microsomes, phenolic glucuronidation was the highest in mice for T4 and in rats for T3 and lowest in monkeys for both T4 and T3. Acyl glucuronidation was highest in humans and lowest in mice for T4 and T3. Phenolic glucuronidation was higher than acyl glucuronidation for T4 in humans; in contrast, the acyl glucuronidation was slightly higher than phenolic glucuronidation for T3. UGT activities were lower toward T3 than T4 in all the species. The LC/MS method was a useful tool in studying glucuronidation of T4 and T3.

Glucuronidation of T4 and T3 may take place on the phenolic hydroxy and carboxyl groups (Fig. 1) in the molecules to give phenolic and acyl glucuronides, respectively (Jemnitz and Vereczkey, 1996). However, there is little documented information about separation of phenolic and acyl glucuronides of thyroid hormone and their formation by UGT isoforms, especially the acyl glucuronides of these two hormones. In the present study, a liquid chromatography/mass spectrometry (LC/MS) method was developed to separate and characterize the phenolic and acyl glucuronides of T4 and T3. This method was used to evaluate formation of phenolic and acyl glucuronides of T4 and T3 by recombinant human UGT isoforms and hepatic microsomes of animals and humans.

View larger version (7K): [in this window] [in a new window]   FIG. 1. Formation of the phenolic glucuronide and acyl glucuronide of T4 (r = I) and T3 (r = H).

	Materials and Methods

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Incubations. Incubations with liver microsomes or recombinant human UGTs consisted of T4 or T3 (100 µM), magnesium chloride (10 mM), microsomes (2 mg/ml) or supersomes (0.5 mg/ml), and UDP-glucuronic acid (UDPGA) (6 mM) in 0.5 ml of 0.1 M Tris-HCl buffer, pH 6.8 or 7.4. Alamethicin (50 µg/mg protein) was mixed with liver microsomes for use in incubations. T4 or T3 in 5 µl of methanol was added to the incubation tubes containing buffer, magnesium chloride solution, alamethicin, and microsomes or UGT isoforms. After mixing, the tubes were preincubated for 3 min in a shaking water bath at 37°C. The reactions were initiated by the addition of 30 µl of 100 mM UDPGA solution in water. Control incubations were conducted under the same conditions without UDPGA or microsomes. All the incubations were performed in duplicate. Reactions were stopped by the addition of 100 µl of ice-cold methanol containing 5% trichloroacetic acid. Fexofenadine in 30 µl of 10 µM solution in methanol was added to each incubation as an internal standard, followed by the addition of 600 µl of methanol. Samples were vortex-mixed. Denatured proteins were separated by centrifugation at 4300 rpm and 4°C for 10 min in a Sorvall (Newton, CT) model T21 super centrifuge. An aliquot of the supernatant was analyzed by LC/MS.

To evaluate the effect of pH on the formation of T4 glucuronides, T4 was incubated with human liver microsomes at pH 6.8 and 7.4 in the presence of UDPGA at 37°C for 5, 10, 15, 20, 30, and 60 min. All the other incubations were conducted at pH 6.8 for 30 min.

Hydrolysis of T4 and T3 Glucuronides. The sample prepared from incubations with human liver microsomes in the presence of UDPGA was dried under a nitrogen stream in a TurboVap LV evaporator (Zymark Corp., Hopkinton, MA). The residue was reconstituted in 1 ml of 0.1 M sodium acetate buffer, pH 5.0. Approximately 560 units of ß-glucuronidase in 100 µl of 0.1 M sodium acetate buffer, pH 5.0, was added to 0.5 ml of the reconstituted solution, and the mixture was incubated at 37°C for 30 min with gentle shaking. The reaction was stopped by the addition of 1 ml of acetonitrile, and the precipitated enzyme was removed by centrifugation. The supernatant was evaporated under nitrogen to remove acetonitrile. An aliquot of the aqueous residue was analyzed by HPLC. A control incubation was prepared under the same conditions but without ß-glucuronidase.

For alkaline hydrolysis, the sample was adjusted with 2N sodium hydroxide to about pH 11 and incubated at 37°C for 60 min. After adjustment with acetic acid to about pH 5, an aliquot was analyzed by HPLC. A control incubation was conducted at pH 6.8. All the samples were analyzed by LC/MS to monitor the glucuronides and the internal standard.

LC/MS. A Waters model 2690 HPLC system (Waters Corp., Milford, MA) with a built-in autosampler and a diode array UV detector was used for analysis. The UV detector was set to monitor 200 to 400 nm. Separations were accomplished on a Luna C18(2) column (150 x 2.0 mm, 5 µm) (Phenomenex, Torrance, CA). The sample chamber in the autosampler was maintained at 4°C, whereas the column was at ambient temperature of about 20°C. The mobile phase consisted of 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B) and was delivered at 0.2 ml/min. The mobile phase B linearly increased from 25% to 65% over the first 5 min, to 85% over 10 min, and then to 95% over 5 min. The first 7 min of flow was diverted away from the mass spectrometer.

The HPLC system was coupled to a Micromass Quattro Ultima triple quadrupole mass spectrometer (Micromass, Inc., Beverly, MA), which was equipped with an electrospray ionization interface and operated in the positive ionization mode. The capillary and cone voltages were set at 4000 and 20 V, respectively. The cone and the desolvation gas flows were 100 and 1100 l/h, respectively. The source block and the desolvation gas temperatures were set at 105 and 200°C, respectively. Mass resolution for multiple reaction monitoring was 15 for MS1 and MS2. The scan mode used selected reaction monitoring with ion transitions of m/z 953.5 to 777.5 for T4 glucuronides, m/z 827.7 to 651.7 for T3 glucuronides, and m/z 502 to 466 for fexofenadine at collision energy of –25 eV. Micromass MassLynx software (version 4.0) was used for collection and analysis of LC/MS data. The peak area ratio of glucuronides to the internal standard was used for comparison of enzyme activities.

To evaluate the limit of detection and linearity of MS response, T4 was incubated with human liver microsomes in the presence of a mixture of [¹⁴C]UDPGA and nonlabeled UDPGA (1:5) as described under Incubations. The phenolic glucuronide was isolated by HPLC using the same conditions described above, except that a 250 x 4.6-mm column was used, and the mobile phase was delivered at 0.6 ml/min. The isolated fractions were concentrated under nitrogen and reconstituted with the control incubation mixture. An aliquot of the supernatant was analyzed by LC/MS after protein precipitation. The amount of T4 glucuronide was calculated by the specific radioactivity of [¹⁴C]UDPGA in the incubation and the radioactivity in the isolated fractions after concentration.

NMR Analysis of T4 Phenolic Glucuronide. T4 phenolic glucuronide was isolated as described in the above section from incubations of T4 with human liver microsomes in the presence of nonradiolabeled UDPGA. The isolated fractions were dried under a nitrogen stream and reconstituted in approximately 160 µl of deuterated methanol (CD₃OD) and transferred to a 3-mm o.d. NMR tube. For the purpose of comparison, a saturated T4 solution was also prepared in CD₃OD. Data were collected on Varian Inova 500 MHz and Bruker Avance 600 MHz spectrometers, each equipped with a 3-mm z-gradient indirect detection probe. One-dimensional proton ¹H NMR data and two-dimensional gradient selected correlation spectroscopy, total correlation spectroscopy, heteronuclear ¹H-¹³C gradient heteronuclear single quanta correlation, and ¹H-¹³C gradient selected heteronuclear multiple bond correlation (Croasmun and Carlson, 1994) NMR data were acquired. All the spectra were referenced to the signal of CHD₂OD at 3.32 ppm in ¹H and 48.1 ppm in ¹³C spectra. Because of limited quantity of metabolite, the ¹³C chemical shifts were extracted from heteronuclear gradient heteronuclear single quanta correlation and gradient selected heteronuclear multiple bond correlation.

View larger version (18K): [in this window] [in a new window]   FIG. 2. T4 glucuronides after incubation without (A) and with (B) ß-glucuronidase.

	Results

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Identification of T4 and T3 Glucuronides. The identities of the T4 glucuronides were elucidated by hydrolysis, LC/MS, and NMR. After hydrolysis with ß-glucuronidase for 30 min, both glucuronide peaks completely disappeared (Fig. 2). After hydrolysis under alkaline conditions for 1 h, the second glucuronide peak completely disappeared, whereas the first glucuronide peak was intact (Fig. 3), suggesting that the first glucuronide was stable and the second glucuronide was labile under alkaline conditions. These findings suggested that the first peak was a phenolic glucuronide and the second peak was an acyl glucuronide of T4.

View larger version (18K): [in this window] [in a new window]   FIG. 3. T4 glucuronides after incubation at pH 6.8 (A) and at pH 11.0 (B).

View larger version (19K): [in this window] [in a new window]   FIG. 4. Product ions of m/z 953.5 for peaks eluted at 13.2 and 14.8 min.

View larger version (25K): [in this window] [in a new window]   FIG. 5. Comparison of the proton NMR spectra of T4 with that of phenolic glucuronide of T4.

View larger version (44K): [in this window] [in a new window]   FIG. 6. Gradient selected heteronuclear multiple bond correlation NMR spectrum of phenolic glucuronide of T4.

T3 glucuronides were identified by LC/MS and hydrolysis. The molecular ions for T3 glucuronides were observed at m/z 827.7, which was 176 Da larger than the molecular ion for T3. Neutral loss of 176 Da from the molecular ions of T3 glucuronides produced m/z 651.7, identical to T3 molecular ion, indicating that the metabolites were T3 glucuronides (data not shown). Similar to T4 glucuronides described above, both T3 glucuronides were hydrolyzed by ß-glucuronidase, whereas only acyl glucuronide was hydrolyzed under alkaline conditions (data not shown).

Effect of pH on the Formation of T4 Glucuronides. Formation of the phenolic and acyl glucuronides of T4 was evaluated at pH 6.8 and 7.4 (Fig. 7). At both pH conditions, formation of the phenolic glucuronide of T4 increased over incubation time up to 60 min. Formation of the acyl glucuronide of T4 reached the highest level at 20 min of incubation at pH 7.4 and 30 min at pH 6.8. Formation of phenolic glucuronide was favored by pH 7.4, whereas formation of the acyl glucuronide was favored by pH 6.8. Therefore, incubations were carried out at pH 6.8 for 30 min in the present study to ensure detection of the acyl glucuronide. Under these conditions, formation of the acyl glucuronide was about 60% higher, whereas formation of the phenolic glucuronide was about 40% lower compared with incubations at pH 7.4.

View larger version (27K): [in this window] [in a new window]   FIG. 7. Effect of pH on the formation of the phenolic and acyl glucuronides of T4. Data represent the average of duplicate experiments.

View larger version (27K): [in this window] [in a new window]   FIG. 8. Glucuronidation of T4 by recombinant human UGT. Data represent the average of duplicate experiments.

View larger version (23K): [in this window] [in a new window]   FIG. 9. Glucuronidation of T3 by recombinant human UGT. Data represent the average of duplicate experiments.

Species Differences in Glucuronidation of T4 and T3. Species differences were observed in glucuronidation of T4 and T3 (Figs. 10 and 11) by liver microsomes. The microsomal UGT activities across species were normalized with P450 contents described under Materials and Methods. Formation of T4 phenolic glucuronide was in the order of mouse > rat, human > dog > monkey, whereas formation of T4 acyl glucuronide was in the order of human > dog > mouse, rat, monkey. Female rats had slightly higher activity than male rats in T4 phenolic glucuronidation. Formation of T3 phenolic glucuronide was observed in the order of rat > mouse > dog > monkey and human, whereas formation of T3 acyl glucuronide was observed in the order of human > dog > rat, monkey > mouse. Female rats had higher activity than male rats in formation of T3 phenolic glucuronide. Humans generated the highest amounts of acyl glucuronides of T4 and T3. In human liver microsomes, phenolic glucuronide was formed in higher amounts than acyl glucuronide for T4; in contrast, the acyl glucuronide was formed in slightly higher amounts than phenolic glucuronide for T3. Humans had the highest ratios of the acyl glucuronide of T4 or T3 to the phenolic glucuronide of T4 or T3. UGT activities toward T3 were lower than those toward T4 in all the species.

View larger version (30K): [in this window] [in a new window]   FIG. 10. Species comparison of T4 glucuronidation. Data represent the average of duplicate experiments.

View larger version (26K): [in this window] [in a new window]   FIG. 11. Species comparison of T3 glucuronidation. Data represent the average of duplicate experiments.

	Discussion

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Formation of the phenolic glucuronides of thyroid hormones has been well studied (Visser et al., 1993a, b; Visser, 1996). Study of the acyl glucuronides of T4 and T3 could be hindered by their low formation and instability. For example, Jemnitz and Vereczkey (1996) reported that T4 acyl glucuronide was completely hydrolyzed at 37°C in an overnight incubation even at pH 4.5. It is well documented that acyl glucuronides are labile and may undergo a number of reactions, including hydrolysis, rearrangement, and covalent binding to proteins, although the reactivity varies largely depending on their structures (Spahn-Langguth and Benet, 1992; Sallustio et al., 2000). Degradation of acyl glucuronides is pH-dependent (Dickinson and King, 1989; Smith and Liu, 1993; Sekikawa et al., 1995; McGurk et al., 1996), and lower than physiological pH conditions have been used to favor the stability of acyl glucuronides (Shipkova et al., 2001). In the present study, a sensitive LC/MS method was developed for the first time to separate and detect the phenolic and acyl glucuronides of T4 and T3, and the structure of the phenolic glucuronide of T4 was unequivocally elucidated by LC/MS and NMR.

Formation of the phenolic and acyl glucuronides of T4 and T3 is catalyzed by multiple human UGT isoforms. UGT1A1, UGT1A3, UGT1A8, UGT1A10, and UGT2B4 are the major isoforms responsible for the formation of the phenolic glucuronide of T4. UGT1A1, UGT1A3, UGT1A8, and UGT1A10 are also the major isoforms catalyzing the formation of the phenolic glucuronide of T3, with lower activity than T4. UGT1A3 was the major isoform catalyzing the formation of the acyl glucuronide of T4 and T3; minor activity was observed with UGT1A1, UGT1A4, and UGT2B7. Our results are consistent with literature reports and offer some new insights into the UGTs involved in glucuronidation of these two compounds. Previous studies have showed that multiple UGT isoforms are involved in the formation of the phenolic glucuronides of T4 and T3 (Visser, 1996). T4 is predominantly glucuronidated in rat liver by both bilirubin and phenol UGTs, whereas T3 is primarily conjugated by androsterone UGT. In the V79 cell lines transfected with cDNA of different human UGTs, Visser et al. (1993a) reported significant conjugation of T4 by bilirubin UGT HP3 (UGT1A1) and the phenol UGT HP4 (UGT1A9). However, the planar phenol UGT isoform HP1 (UGT1A6) had little or no glucuronidation of iodothyronines. Similar findings were also reported by Findlay et al. (2000) with human liver microsomes. Vansell and Klaassen (2002) reported in rat hepatocytes that T4 glucuronidation was catalyzed by UGT1A1 and UGT1A6, whereas conjugation of T3 was catalyzed by UGT2B2. That activity was detected with multiple human UGT isoforms in the present study might be attributed to overlapping substrate specificity for UGTs (Radominska-Pandya et al., 1999). Formation of the acyl glucuronides of thyroid hormones by UGT isoforms has not previously been reported. However, at least six human UGTs—UGT1A1, UGT1A3, UGT1A8, UGT1A9, UGT1A10, and UGT2B7—have been identified catalyzing the glucuronidation of carboxylic drugs (Gall et al., 1999; Mackenzie, 2000; Sallustio et al., 2000). These UGTs also catalyze the glucuronidation of a number of noncarboxylic acid compounds, including both endogenous and xenobiotic chemicals, and have overlapping but different substrate specificities (Sallustio et al., 2000). Significant differences in relative specificities toward the same carboxylic acid substrates have been shown (Sallustio et al., 2000). Formation of acyl glucuronides of T4 and T3 by UGT1A8, UGT1A9, or UGT1A10 was not observed in the present study. UGT1A4, which conjugates a structurally diverse group of compounds including drugs, xenobiotics, hydroxysteroids, and particularly primary, secondary, and tertiary amine-containing compounds (Green et al., 1995; Green and Tephly, 1996), yielded the acyl but not the phenolic glucuronide of T4 and T3. Because UGT1A7, UGT1A8, and UGT1A10 are extrahepatic enzymes (Strassburg et al., 1997, 1999), UGT1A1 and UGT1A3, which are expressed in human liver, may play major roles in T4 glucuronidation in humans.

Species differences in glucuronidation of T4 and T3 were also observed under the experimental conditions. Mice have much higher activity toward T4 than any other species, whereas rats have the highest activity toward T3 among all the species examined. Female rats show higher T3 UGT activity than male rats. The yields of T3 phenolic glucuronide were lower, whereas the acyl glucuronides of both T4 and T3 were formed in higher amounts in humans among the species examined. Species and sex differences in UGT activities toward thyroid hormones have not been well documented, although species differences in response to chemical inducers have been reported (Craft et al., 2002; Kato et al., 2003).

In summary, a novel LC/MS method was developed to separate and detect the phenolic and acyl glucuronides of T4 and T3. This method was used to evaluate a number of UGT isoforms for formation of the phenolic and acyl glucuronides of T4 and T3 to study species differences in T4 and T3 glucuronidation in liver microsomes of animals and humans. Among the 11 UGT isoforms examined, UGT1A1, UGT1A3, UGT1A8, UGT1A10, and UGT2B4 were the major isoforms responsible for the formation of T4 phenolic glucuronide, whereas UGT1A1, UGT1A3, UGT1A8, and UGT1A10 catalyzed the formation of T3 phenolic glucuronide. UGT1A3 was the major isoform responsible for the formation of the acyl glucuronides of T4 and T3. The activity in formation of T4 and T3 phenolic glucuronide was lower, whereas the activity in formation of T4 and T3 acyl glucuronides was higher in human liver microsomes than in other species.

	Acknowledgments

 
We thank Li Shen for her suggestions and help in the literature search, Lisa Nogle for her help in the NMR analysis, and Theresa Hultin and JoAnn Scatina for supporting this research and reviewing the manuscript.

	Footnotes

 
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.107.016972.

ABBREVIATIONS: T4, thyroxine; T3, triiodothyronine; UGT, UDP-glucuronosyltransferase; LC/MS, liquid chromatography/mass spectrometry; HPLC, high-performance liquid chromatography; UDPGA, UDP-glucuronic acid.

Address correspondence to: Zeen Tong, Drug Safety and Metabolism, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426. E-mail: tongz{at}wyeth.com

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This Article Abstract Full Text (PDF) All Versions of this Article: dmd.107.016972v1 35/12/2203    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Tong, Z. Articles by Chandrasekaran, A. Search for Related Content PubMed PubMed Citation Articles by Tong, Z. Articles by Chandrasekaran, A.