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Endocrinology 149(6):3037–3045 Copyright © 2008 by The Endocrine Society doi: 10.1210/en.2007-1678
Thyronamines Are Isozyme-Specific Substrates of Deiodinases S. Piehl, T. Heberer, G. Balizs, T. S. Scanlan, R. Smits, B. Koksch, and J. Ko¨hrle Institut fu¨r Experimentelle Endokrinologie und Endokrinologisches Forschungszentrum der Charite´ EnForCe´ (S.P., J.K.), Charite´–Universita¨tsmedizin Berlin, Campus Virchowklinikum, D-13353 Berlin, Germany; Federal Institute for Risk Assessment (T.H., G.B.), D-12277 Berlin, Germany; Department of Physiology and Pharmacology (T.S.S.), Oregon Health and Science University, Portland, Oregon 97239; Department of Chemistry and Biochemistry–Organic Chemistry (R.S., B.K.), Free University of Berlin, D-14195 Berlin, Germany 3-Iodothyronamine (3-T1AM) and thyronamine (T0AM) are novel endogenous signaling molecules that exhibit great structural similarity to thyroid hormones but apparently antagonize classical thyroid hormone (T3) actions. Their proposed biosynthesis from thyroid hormones would require decarboxylation and more or less extensive deiodination. Deiodinases (Dio1, Dio2, and Dio3) catalyze the removal of iodine from their substrates. Because a role of deiodinases in thyronamine biosynthesis requires their ability to accept thyronamines as substrates, we investigated whether thyronamines are converted by deiodinases. Thyronamines were incubated with isozyme-specific deiodinase preparations. Deiodination products were analyzed using a newly established method applying liquid chromatography and tandem mass spectrometry (LC-MS/MS). Phenolic ring deiodinations of 3,3ⴕ,5ⴕ-triiodothyronamine (rT3AM), 3ⴕ,5ⴕdiiodothyronamine (3ⴕ,5ⴕ-T2AM), and 3,3ⴕ-diiodothyronamine (3,3ⴕ-T2AM) as well as tyrosyl ring deiodinations of 3,5,3ⴕ-triiodo-
thyronamine (T3AM) and 3,5-diiodothyronamine (3,5-T2AM) were observed with Dio1. These reactions were completely inhibited by the Dio1-specific inhibitor 6n-propyl-2-thiouracil (PTU). Dio2 containing preparations also deiodinated rT3AM and 3ⴕ,5ⴕ-T2AM at the phenolic rings but in a PTU-insensitive fashion. All thyronamines with tyrosyl ring iodine atoms were 5(3)-deiodinated by Dio3-containing preparations. In functional competition assays, the newly identified thyronamine substrates inhibited an established iodothyronine deiodination reaction. By contrast, thyronamines that had been excluded as deiodinase substrates in LC-MS/MS experiments failed to show any effect in the competition assays, thus verifying the former results. These data support a role for deiodinases in thyronamine biosynthesis and contribute to confining the biosynthetic pathways for 3-T1AM and T0AM. (Endocrinology 149: 3037–3045, 2008)
T
nes (e.g. T4 or T3), decarboxylation of the -alanine side chain would be required for their biosynthesis. However, so far, no iodothyronine-decarboxylating enzyme has been identified (4). If the putative decarboxylating enzyme was converting only iodothyronines with higher iodine content, deiodinases would be directly required to complete 3-T1AM and T0AM biosynthesis by removing at least two to three iodine atoms. A role of deiodinases in thyronamine biosynthesis presupposes their ability to accept thyronamines as substrates. So far, the three deiodinase isozymes (Dio1, Dio2, and Dio3) have been described to catalyze the sequential reductive removal of iodine from iodothyronines and various iodothyronine metabolites thus controlling the bioavailability of thyroid hormones (5). Although Dio1 exhibits both phenolic and tyrosyl ring deiodination activity (Fig. 1), Dio2 and Dio3 are more specific with respect to the position of the iodine removed. Dio2 catalyzes only deiodinations of the phenolic ring, e.g. the conversion of the prohormone T4 to active T3, whereas Dio3 catalyzes only deiodinations of the tyrosyl ring, e.g. the conversion of T4 to inactive rT3 (5). In this study, the complete panel of thyronamine deiodination reactions was investigated systematically. Because the various thyronamines differ only regarding the number or the position of the iodine atoms, their distinction by immunological methods has been hampered so far. Therefore, a novel liquid chromatography and tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous detection of all thyronamines in the same sample. The
HYRONAMINES (FIG. 1) ARE a novel class of endogenous signaling compounds. In terms of structure, they differ from thyroid hormone l-thyroxine (T4) and deiodinated thyroid hormone derivatives only concerning the absence of the carboxylate group of the -alanine side chain. Thyronamine nomenclature follows the rules applied for thyronines (TxAM with x indicating the number of iodine atoms per molecule). So far, only two representatives of thyronamines, namely 3-iodothyronamine (3-T1AM) and thyronamine (T0AM), have been detected in vivo in various species (1). Although their physiological roles still remain elusive, 3-T1AM and T0AM have exhibited short-term hypothermic, negative chronotropic, and negative inotropic effects that are opposite in direction to the actions of the classical active thyroid hormone T3 (1–3). Thus, thyronamines were suggested to be derivatives of thyroid hormones that might serve to fine-tune or even antagonize thyroid hormone effects. Yet the pathways of thyronamine biosynthesis are still unknown. If thyronamines were derivatives of iodothyroniFirst Published Online March 13, 2008 Abbreviations: Dio1, Deiodinase 1; DTT, dithiothreitol; LC-MS/MS, liquid chromatography and tandem mass spectrometry; PTU, 6n-propyl-2-thiouracil; SULT, sulfotransferase. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.
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Piehl et al. • Thyronamines Are Substrates of Deiodinases
vol) fetal calf serum (Biochrom) and 100 nm sodium selenite for optimal expression of deiodinase selenoproteins (8). At confluence, HEK293 and HepG2 cells were directly harvested for deiodinase assays. By contrast, confluent MSTO-211H cells were serum starved overnight and then incubated with 10 m forskolin and 700 nm MG-132 for 6 h to stimulate the endogenous Dio2 enzyme activity. Likewise, confluent and serumstarved ECC-1 cells were treated with 100 nm phorbol-12-myristate-13acetate for 6 h to increase the endogenous Dio3 enzymatic activity.
LC-MS/MS detection of thyronamines
FIG. 1. Structure and nomenclature of thyronamines.
experiments revealed that all three deiodinases catalyze thyronamine deiodination reactions with each isozyme exhibiting a unique substrate specificity. Materials and Methods Chemicals Thyronamines were synthesized as described before (6) as was deuterated 3-T1AM (3-T1AM-d4) (7). rT3 and T3 were kindly provided by Dr. Rudy Thoma (Formula, Berlin, Germany). l-[5⬘-125I]rT3 was purchased from PerkinElmer (Ju¨gesheim, Germany). Forskolin and phorbol-12myristate-13-acetate were obtained from Sigma-Aldrich (Steinheim, Germany). The proteasome inhibitor MG-132 was purchased from Biomol (Plymouth Meeting, PA).
Animals Livers from two euthyroid, adult, male C57BL/6 wild-type mice were kindly provided by Dr. Ulrich Schweizer (Institute of Experimental Endocrinology, Charite´, Berlin).
Cell culture The human ECC-1 endometrium carcinoma cell line was kindly provided by Dr. Monique Kester (Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands). HepG2 and ECC-1 cells were propagated in DMEM-F12 medium (Invitrogen, Karlsruhe, Germany). HEK293 cells were cultured in DMEM (Biochrom, Berlin, Germany), and MSTO-211H cells were grown in RPMI 1640 medium (Invitrogen). All cell culture media were supplemented with 10% (vol/
LC-MS/MS analyses were performed using an Agilent 1100 HPLC system (Agilent, Waldbronn, Germany) and an API 365 triple-quadrupole tandem mass spectrometer (Applied Biosystems, Darmstadt, Germany) equipped with TurboIonSpray interface. The detection was performed using positive electrospray ionization (ESI⫹) in the selected reaction monitoring (SRM) mode. Data processing was performed using Bio Analyst version 1.3.1 software (Applied Biosystems). All compound-specific mass spectrometric working parameters were optimized by directly injecting thyronamine standard solutions (10 g/ ml) at a flow rate of 10 l/min into the mass spectrometer and were summarized in Table 1. Because all thyronamines showed the loss of ammonia in their tandem mass spectra, the transition (M⫹H)⫹ 3 (M⫹H-NH3)⫹ was used for their detection by MS/MS. Chromatographic separation of all thyronamines was achieved using a Synergi Polar-RP 80-Å column (150 ⫻ 2 mm; Phenomenex, Aschaffenburg, Germany) with a 0.3 ml/min gradient elution program (Fig. 2). The optimized elution parameters were mobile phase A (water-acetonitrile-acetic acid, 95:5:0.6) and mobile phase B (water-acetonitrile-acetic acid, 5:95:0.6): 0 –1 min, 10% B; 1–15 min, 10 –50% B; 15–20 min, 50% B; 20 –24 min, 50 –10% B; and 25–29 min, 10% B. The remaining mass spectrometric working parameters were source temperature, 300 C; dwell time, 90 msec; Q1 peak width, 0.7; and Q3 peak width, 0.7. The LC-MS/MS method was validated for the detection of thyronamines from extracted deiodinase reaction matrices according to internationally accepted recommendations (9, 10). The method was selective for all thyronamines, and no matrix effects were observed. It was linear over a wide range of thyronamine concentrations in deiodinase reactions (100 pm to 7.5 m). Linear regression of calibrator peak area ratios (i.e. TxAM/T1AM-d4 vs. moles TxAM) yielded coefficients of determination (r2) greater than 0.997. Linearity experiments were also used to verify T1AM-d4 as an appropriate internal standard for all thyronamines. The limits of detection and lower limits of quantitation were calculated using a signal-to-noise ratio of at least 3:1 and 10:1, respectively. The limits of detection of the analyte concentrations in deiodinase reaction matrices were 100 pm for T0AM and mono- and diiodothyronamines and 1 nm for triiodothyronamines and T4AM. The lower limits of quantitation of the analyte concentrations in deiodinase reactions were 250 pm for T0AM and mono- and diiodothyronamines and 2.5 nm for triiodothyronamines and T4AM. The assay stability was verified by determining the following parameters: intra- and interassay precision of retention times were less than 2.14 sec and less than 2.18 sec, respectively. Intra- and interassay precision analyte concentrations ranged from 5.0 – 8.3% and 5.6 –11.0%, respectively. Thus, precision data were within the required limits of 15% coefficient of variation at all analyte concentrations studied (9, 10). Intra-
TABLE 1. Compound-specific mass spectrometer working parameters for the detection of thyronamines by LC-MS/MS Compound
(m/z) Q1
(m/z) Q3
DP (V)
FP (V)
EP (V)
CEP (V)
CE (V)
T0AM 3-T1AM , 3⬘-T1AM 3-T1AM-d4 3,5-T2AM 3,3⬘-T2AM 3⬘,5⬘-T2AM T3AM, rT3AM T4AM
230.3 356.2 360.2 482.1
213.3 339.2 343.2 465.1
3.0 10.0 10.7 12.0
144.0 138.0 169.0 177.0
5.0 6.0 6.0 7.6
94.9 85.0 84.6 74.9
15.6 15.0 15.0 16.0
CXP (V)
5.0 8.0 8.1 9.0
608.0 733.9
591.0 716.9
19.0 32.0
196.0 235.0
7.7 9.4
64.9 54.9
20.1 25.0
12.0 15.0
CE, Collision energy; CEP, collision cell entrance potential; CXP, collision cell exit potential; DP, declustering potential; FP, focusing potential; EP, entrance potential; (m/z) Q1, mass to charge ratio of the mother ion in the first quadrupole; (m/z) Q3, mass to charge ratio of the most intensive daughter ion in the third quadrupole.
Piehl et al. • Thyronamines Are Substrates of Deiodinases
Endocrinology, June 2008, 149(6):3037–3045
FIG. 2. Representative LC-MS/MS chromatogram of thyronamine standard solutions. Thirty picomoles of T0AM, 3-T1AM, 3⬘-T1AM, 3,5-T2AM, and 3,3⬘-T2AM were injected each. Due to the lower intensity of T3AM, rT3AM and T4AM in this assay, 150 pmol of these compounds had to be injected to obtain peaks that were high enough to present them together with T0AM and mono- and diiodothyronamines in one chromatogram. T1AM-d4 (internal standard) and 3⬘,5⬘-T2AM were not injected because their retention times (13.0 and 15.6 min, respectively) were almost identical to that of 3-T1AM and 3,3⬘-T2AM, respectively (A). Identification of thyronamines and compound specific retention times (rt) (B). cps, Counts per second. and interassay bias of analyte concentration ranged from 3.3–10.3% and 6.1–12.4%. Thus, bias data were also within the acceptance range of ⫾15% of the nominal values at all analyte concentrations (9, 10).
LC-MS/MS-based deiodinase assays Mouse liver membrane fractions were prepared as described before (11). To generate cell line-derived deiodinase preparations, confluent cells were washed twice with ice-cold 1⫻ PBS at pH 7.4 and harvested by scraping into a homogenization buffer containing 250 mm sucrose, 20 mm HEPES, 1 mm EDTA, and 1 mm dithiothreitol (DTT). HepG2 and MSTO-211H cells were harvested into a homogenization buffer at pH 7.4, whereas ECC-1 cells were scraped into a homogenization buffer at pH 8.0 (Table 2). After sonication, the protein concentrations were determined using Bradford assay (12). Samples containing appropriate amounts of protein (Table 2) were prepared in the respective homogenization buffer and stored at ⫺20 C until use. Aliquots of cell line lysates and mouse liver membrane fractions were thawed on ice. Deiodination reactions were started by incubating the aliquots at 37 C in 1.5 vol potassium phosphate buffer containing 167 mm K2HPO4/KH2PO4, 1.67 m Na2EDTA䡠H2O, 273 m NaOH, and different concentrations of DTT (Table 2). Thyronamines, rT3 (which served as a positive control substrate for Dio1 and Dio2), or T3 (which served as a positive control substrate for Dio3) were added to the deiodinase reactions at various final concentrations as listed in Table 2. Each substrate was assayed at all concentrations in at least three separate experiments. Within one such experiment, each substrate concentration was analyzed in three replicate reactions containing 1 mm 6n-propyl-2-thiouracil
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(PTU) and three replicate reactions devoid of PTU. As an exception to this, all reactions with Dio3-containing ECC-1 lysates were performed in the presence of 1 mm PTU to inhibit the weak Dio1 activity found in this cell line. The enzymatic reactions were stopped by adding 0.1 vol 100% (vol/ vol) acetic acid. Subsequently, 4 pmol 3-T1AM-d4 was added to each vial to serve as internal standard. The reaction mixtures were incubated at 37 C for 60 min. Proteins were precipitated by adding 3 vol ice-cold acetone and incubating at ⫺20 C for 15 min. After centrifugation at 14,000 ⫻ g and 4 C for 5 min, the supernatants were transferred into new Eppendorf tubes, acidified with 0.002 vol 30% (vol/vol) HCl, washed twice with 1 vol cyclohexane, and then subjected to three subsequent extraction cycles with 1.5 vol ethyl acetate. The organic layers were combined and evaporated to dryness at 45 C. The residues were redissolved in 30 l H2O-methanol-acetic acid (90:10:1) and stored at ⫺20 C until analysis of the deiodination products by LC-MS/MS. The injection volumes were 10 l with analytes dissolved in the mobile phase used for LC-MS/MS analysis. The extraction efficiencies of thyronamines from deiodinase reaction matrices were routinely controlled in every run. Recovery rates were not significantly influenced by the type of cell line lysate, by the pH of the deiodinase reaction, or by PTU addition. Furthermore, in every experiment, calibration curves for each analyte were recorded taking the compound specific recovery rates into consideration.
Comparison of LC-MS/MS-based deiodinase assays and 125 ⫺ I release assays To validate the performance of the novel LC-MS/MS method in deiodinase assays, the apparent Km and Vmax values of selected iodothyronine deiodination reactions were determined by LC-MS/MS assays and compared with the values obtained from classical radioactive 125 ⫺ I release assays. The following substrates were used: 50 nm to 4.4 m rT3 for Dio1, 10 –50 nm rT3 for Dio2, and 10 –100 nm T3 for Dio3. All reactions were stopped after 30 min. The specific activities were expressed as moles 3,3⬘-T2 per milligram protein per minute and moles 125 ⫺ I released per milligram protein per minute for LC-MS/MS and 125I⫺ release experiments, respectively. The apparent Km and Vmax values were determined by fitting the enzyme kinetic data to the MichaelisMenten equation by means of nonlinear regression using GraphPad Prism Software version 4.00 (GraphPad, San Diego, CA). The values reported for apparent Km and Vmax are from three separate experiments performed in triplicate and are presented as mean ⫾ sd. For Dio1 and Dio2, the values obtained by LC-MS/MS-based deiodinase assays and 125 ⫺ I release assays did not differ significantly as calculated by Wilcoxon test (data not shown). For Dio3 from ECC-1 lysates, the specific activities measured in LC-MS/MS-based deiodinase assays were in line with those obtained by a radiochemical, HPLC-based assay (13).
Apparent Km and Vmax of selected thyronamine deiodination reactions The apparent Km and Vmax values of selected thyronamine deiodination reactions were measured by LC-MS/MS-based deiodinase assays using 10 nm–5 m thyronamine substrate and an incubation time of 30 min. The specific activities were expressed as moles deiodinated product per milligram protein per minute. The values reported for apparent Km and Vmax were calculated as described above and are presented as mean ⫾ sd from three separate experiments performed in triplicate.
TABLE 2. Design of LC-MS/MS-based deiodinase assays
pH of homogenization buffer Amount of protein per reaction (g) pH of potassium phosphate buffer 关DTT兴 in potassium phosphate buffer (mM) Final 关substrate兴 (M) Incubation time (min) a
HepG2
Mouse liver
MSTO-211H
ECC-1
HEK293
7.4 300 6.8 33 0.05–20 30 –120
7.4 40 6.8 33 0.05–20 15–120
7.4 2000 6.8 33 0.01– 0.5 30 –120
8.0 600 8.0 83 0.01–5 30 –120
7.4 or 8.0a 40 –2000a 6.8 or 8.0a 33 or 83a 0.01–20a 15–120a
Adapted to the conditions used in the respective series of samples.
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Dio1 and Dio2 assays based on
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Piehl et al. • Thyronamines Are Substrates of Deiodinases
I-release (14)
Mouse liver membrane fractions and cell line-derived deiodinase preparations were generated as described for LC-MS/MS-based deiodinase assays (Table 2) except that different amounts of protein (micrograms per reaction) were used (HepG2, 50; mouse liver membrane fractions, 20; MSTO-211H, 150; ECC-1, 300; HEK293, 20 –300). Aliquots of cell line lysates and mouse liver membrane fractions were thawed on ice. Deiodination reactions were started by incubating the aliquots at 37 C in 1.5 vol potassium phosphate buffer at pH 6.8 containing 167 mm K2HPO4/KH2PO4, 1.67 m Na2EDTA䡠H2O, 273 m NaOH, 33 mm DTT, 500 cpm/l [5⬘-125I]rT3, and various concentrations of unlabeled rT3 (Table. 2). Each sample was analyzed in three separate experiments. Within one such experiment, each sample was analyzed in three replicate reactions containing 1 mm PTU and three replicate reactions devoid of PTU. Reactions were stopped by adding 0.5 vol 10% (wt/vol) BSA and 3 vol 10% (wt/vol) trichloroacetic acid. After centrifugation, the supernatants were analyzed for 125I⫺ content using acetic acid-equilibrated Dowex 50W-X2 ion-exchange resins. The 125I⫺ released in samples containing PTU was attributed to Dio2, whereas the difference in 125I⫺ release between aliquots with and without PTU was used to calculate Dio1 activity.
Competition assays 125 ⫺ I release assays were used to analyze the effect of thyronamines on the phenolic ring deiodination of rT3 by Dio1. Mouse liver membrane fractions were incubated for 15 min with 0.2– 4.4 m rT3 and 0.1–10.0 m thyronamine at each rT3 concentration used. The values reported for apparent Km and Vmax were determined as described above and are presented as mean ⫾ sd from three separate experiments performed in triplicate. Linear regression lines from EadieHofstee plots are used to display the effects of thyronamines on the Km (represented by slope) and Vmax (represented by y-intercept) of the phenolic ring deiodination of rT3 by Dio1 (see Fig. 4, left panel). Ki values were determined by Dixon plots and linear regression analysis (see Fig. 4, right panel). The values reported for Ki are from three separate experiments performed in triplicate and are presented as mean ⫾ sd.
Statistical analyses of competition assays Statistical analyses were performed using GraphPad Prism Software version 4.00. The effect of thyronamines on the apparent Km and Vmax of the conversion of rT3 by Dio1 from mouse liver membrane fractions was analyzed using Friedman test followed by Dunn’s post test. P ⬍ 0.05 was considered significant.
Results Validation of the isozyme specificity of the cell line-derived deiodinase preparations
The following human cell lines were selected as wellestablished sources of endogenous high specific activities of
the respective deiodinase isozyme: HepG2 for Dio1 (13, 15), MSTO-211H for Dio2 (16), and ECC-1 for Dio3 (13). Still, to verify the exclusive functional expression of the respective isozyme in each cell line lysate, the enzymatic activities of the three deiodinase isoforms were analyzed in each lysate using LC-MS/MS. In HepG2 lysates, high specific Dio1 activity but no Dio2 and Dio3 activities were detected (Table 3). Accordingly, HepG2 lysates represented an isozyme-specific preparation of Dio1 and were used to study the ability of Dio1 to accept thyronamines as substrates. In MSTO-211H lysates, only PTU-insensitive Dio2 enzymatic activity was detected (Table 3). Thus, MSTO-211H lysates represented an isozyme-specific preparation of Dio2. In ECC-1 lysates, Dio3 was detected at high specific activities (Table 3). Because also a minimal Dio1 activity was measured (specific activity of 23 ⫾ 3 fmol 3,3⬘-T2/mg protein䡠min at a final rT3 concentration of 1 m), 1 mm PTU was added to each reaction catalyzed by ECC-1 lysates to efficiently inhibit the Dio1 activity. Lysates of HEK293 cells served as deiodinase-deficient negative control preparations because no deiodinase activity was measured (Table 3). Dio1-containing HepG2 lysates deiodinate rT3AM and 3⬘,5⬘-T2AM at the phenolic rings
In positive control experiments, HepG2 lysates were incubated as described in Table 2 using rT3 as a well-defined substrate of Dio1. rT3 was readily converted into its phenolic ring deiodination product 3,3⬘-T2 (Fig. 3A). This conversion was completely inhibited by 1 mm PTU. In negative control experiments using deiodinase-deficient HEK293 lysates or HepG2 lysates that had been heat-inactivated before, no deiodination of rT3 was observed (data not shown). To test the ability of Dio1 from HepG2 cells to accept thyronamines as substrates, HepG2 lysates were incubated as described in Table 2 using various concentrations of thyronamines as substrates. Both rT3AM and 3⬘,5⬘-T2AM were readily deiodinated at their phenolic rings yielding 3,3⬘T2AM and 3⬘-T1AM, respectively, at all substrate concentrations tested (Fig. 3, B and C). Because both reactions were sensitive to PTU inhibition, they were unambiguously catalyzed by Dio1. No conversions of rT3AM and 3⬘,5⬘-T2AM were observed in negative control experiments performed with HEK293 lysates or heat-inactivated HepG2 lysates (data
TABLE 3. Deiodinase isozyme specificity of cell line lysates as measured by LC-MS/MS using selected iodothyronines as substrates
Dio1 Km (M rT3) Vmax (pmol 3,3⬘-T2/mg protein䡠min) Dio2 Km (M rT3) Vmax (pmol 3,3⬘-T2/mg protein䡠min) Dio3 Km (M T3) Vmax (pmol 3,3⬘-T2/mg protein䡠min)
HepG2
MSTO-211H
ECC-1
HEK293
0.59 ⫾ 0.03 2.2 ⫾ 0.06
ND
see text
ND
ND
0.005 ⫾ 0.001 0.130 ⫾ 0.026
ND
ND
ND
ND
0.006 ⫾ 0.002 2.9 ⫾ 0.2
ND
The apparent Km and Vmax values were determined by LC-MS/MS-based deiodinase assays using an incubation time of 30 min and the following substrates: 50 nM to 4.4 M rT3 for Dio1, 10 –50 nM rT3 for Dio2, and 10 –100 nM T3 for Dio1. The values represent the mean ⫾ SD from three separate experiments performed in triplicate. ND, Not detected.
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FIG. 3. LC-MS/MS-based deiodinase assays. In each reaction presented here, 500 nM substrate was used. Each chromatogram represents three separate experiments performed in triplicate. rT3 (A) as well as rT3AM and 3⬘,5⬘-T2AM (B and C) were deiodinated at the phenolic rings by HepG2 lysates in a PTU-sensitive fashion. rT3AM and 3⬘,5⬘-T2AM were deiodinated at the phenolic rings in a PTU-insensitive way by Dio2-containing MSTO-211H lysates (D and E). The positive control substrate T3 was deiodinated at the tyrosyl ring by Dio3 from ECC-1 lysates yielding 3⬘,5⬘-T2 (F). ECC-1 lysates also deiodinated T4AM, rT3AM, T3AM (G–I), 3,3⬘-T2AM, 3,5-T2AM (data not shown), and 3-T1AM (J) at the tyrosyl rings. cps, Counts per second; iS, Internal standard (3-T1AM-d4).
not shown). All other thyronamines were not deiodinated by HepG2 cell lysates under these conditions and at the substrate concentrations tested. Dio2-containing MSTO-211H lysates also deiodinate rT3AM and 3⬘,5⬘-T2AM at the phenolic rings
In positive control experiments, rT3 was deiodinated at the phenolic ring by MSTO-211H lysates yielding 3,3⬘-T2 (data not shown). In contrast to HepG2 lysates, this deiodination was not sensitive to PTU, which is consistent with the known inhibition characteristics of Dio2. Of all thyronamines, only rT3AM and 3⬘,5⬘-T2AM were converted by Dio2 from MSTO211H lysates yielding 3,3⬘-T2AM and 3⬘-T1AM, respectively (Fig. 3, D and E). Dio3-containing ECC-1 lysates deiodinate all thyronamines with iodine atoms in the tyrosyl ring
To inhibit the weak Dio1 enzymatic activity that had been found in ECC-1 cells, 1 mm PTU was added to all incubations. In positive control experiments, ECC-1 lysates were incubated with T3, which represents the preferred substrate of Dio3 (5). T3 was sequentially deiodinated into its tyrosyl ring deiodination products 3,3⬘-T2 and 3⬘-T1 (Fig. 3F). Because this reaction proceeded in the presence of PTU, it was clearly catalyzed by Dio3. All thyronamines with iodine atoms in the tyrosyl ring were deiodinated at their tyrosyl rings (Fig. 3, G–J, illustrating the respective deiodination reactions of T4AM, rT3AM, T3AM, and 3-T1AM). By contrast, those thy-
ronamines without tyrosyl ring iodine, namely 3⬘,5⬘-T2AM and 3⬘-T1AM, were not deiodinated (data not shown). Apparent Km and Vmax of selected thyronamine deiodination reactions
To characterize the kinetic properties of thyronamine deiodinations, the apparent Km and Vmax of selected thyronamine deiodination reactions were measured and compared with that of the respective iodothyronine. Accordingly, the phenolic ring deiodination of rT3AM by Dio1 from HepG2 and by Dio2 from MSTO-211H lysates was compared with that of rT3. With both deiodinase preparations, rT3AM was deiodinated at the phenolic ring at apparent Km values similar to those observed with rT3 but at lower Vmax values compared with rT3 (compare Table 3 with Table 4). Likewise, the tyrosyl ring deiodination of T3AM by Dio3 from ECC-1 lysates was characterized by a similar apparent Km but by a lower apparent Vmax when compared with T3 (compare Table 3 with Table 4). Finally, the apparent Km and Vmax of the 3-deiodination of 3-T1AM to T0AM by Dio3 from ECC-1 lysates was studied, because this reaction might directly account for the biosynthesis of T0AM in vivo. The reaction was catalyzed at an apparent Km of 1.2 ⫾ 0.3 m and an apparent Vmax of 2.4 ⫾ 0.06 pmol T0AM/mg protein䡠min. By contrast, 3-iodothyronine (3-T1), which represents the corresponding iodothyronine, was deiodinated at the tyrosyl ring at an apparent Km of 1.6 ⫾ 0.09 m and an apparent Vmax of 0.089 ⫾ 0.05 pmol T0/mg protein䡠min. Hence, 3-T1AM displayed a higher
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TABLE 4. Apparent Km and Vmax of selected TAM deiodination reactions as measured by LC-MS/MS Deiodinase isozyme
Dio1 Dio2 Dio3 Dio3
Deiodinase preparation
Substrate
Km (M substrate)
Vmax (pmol deiodinated product/mg protein䡠min)
HepG2 MSTO-211H ECC-1 ECC-1
rT3AM rT3AM T3AM 3-T1AM
0.63 ⫾ 0.05 0.006 ⫾ 0.002 0.017 ⫾ 0.004 1.2 ⫾ 0.05
1.5 ⫾ 0.08 0.088 ⫾ 0.012 1.9 ⫾ 0.2 2.4 ⫾ 0.06
The apparent Km and Vmax values were determined by LC-MS/MS-based deiodinase assays using an incubation time of 30 min and 10 nM to 5 M of the indicated thyronamine as substrate. The values represent the mean ⫾ SD from three separate experiments performed in triplicate.
Vmax/Km ratio and thus represented a better substrate of Dio3 from ECC-1 lysates than 3-T1. Thyronamines identified as Dio1 substrates inhibit the Dio1-catalyzed 5⬘-deiodination of rT3
To substantiate the newly identified thyronamine deiodination reactions, we studied which thyronamines would interfere with an established iodothyronine deiodination reaction. Those thyronamines that had been identified as deiodinase substrates in the LC-MS/MS assays were expected to compete with a classical iodothyronine substrate in a deiodinase-mediated catalyzed reaction. By contrast, those thyronamines that were excluded as deiodinase substrates in the LC-MS/MS experiments were not expected to do so. In the following studies, liver membrane fractions from male C57BL/6 wild-type mice were used as an exemplary deiodinase preparation for several reasons. First, high specific Dio1 activity but no Dio2 and Dio3 activities were detected in mouse liver membrane fractions using both classical 125 ⫺ I release assays and LC-MS/MS-based deiodination assays (data not shown). Thus, mouse liver membrane fractions served as a rich and isozyme-specific source of Dio1. Second, mouse liver membrane fractions represented a Dio1 preparation of high physiological and ex vivo relevance. Finally, both phenolic and tyrosyl ring thyronamine deiodination reactions were identified for this enzyme preparation in preliminary LC-MS/MS studies. In line with HepG2 lysates, Dio1 from mouse liver membrane fractions catalyzed phenolic ring deiodinations of rT3AM and 3⬘,5⬘-T2AM yielding 3,3⬘-T2AM and 3⬘-T1AM, respectively (data not shown). Furthermore, mouse liver membrane fractions deiodinated 3,3⬘T2AM at the phenolic ring yielding 3-T1AM and catalyzed weak tyrosyl ring deiodinations of T3AM and 3,5-T2AM producing 3,3⬘-T2AM and 3-T1AM, respectively (data not shown). Because these reactions were all completely inhib-
ited by the addition of 1 mm PTU, they were unambiguously catalyzed by Dio1. To substantiate the identification of those thyronamine substrates of Dio1 in a functional assay, mouse liver membrane fractions were subjected to 125I⫺ release assays using rT3 as substrate and increasing concentrations of the various thyronamines as inhibitors. rT3 was chosen because it represents the preferred substrate of Dio1 (5). In line with previous reports (17), rT3 was deiodinated at the phenolic ring by Dio1 from mouse liver membrane fractions following Michaelis-Menten kinetics with an apparent Km of 0.43 ⫾ 0.023 m and apparent Vmax of 142.7 ⫾ 2.7 pmol 125I⫺ released/mg protein䡠min (Table 5). rT3AM, 3⬘,5⬘-T2AM, and 3,3⬘-T2AM, which had been identified as phenolic ring substrates of Dio1 from mouse liver membrane fractions in LC-MS/MS experiments, inhibited the 5⬘-deiodination of rT3. Both 3⬘,5⬘-T2AM and 3,3⬘-T2AM led to a significant increase in Km as determined by Friedman test followed by Dunn’s post test (Fig. 4A for 3⬘,5⬘-T2AM and Table 5). Moreover, 3⬘,5⬘-T2AM and 3,3⬘-T2AM caused slight decreases in Vmax, which reached statistical significance. Therefore, 3⬘,5⬘T2AM and 3,3⬘-T2AM acted as mixed inhibitors, with competitive and noncompetitive elements. Dixon plots revealed Ki values of 2.6 ⫾ 0.4 m and 4.8 ⫾ 0.1 m for 3⬘,5⬘-T2AM and 3,3⬘-T2AM, respectively (Table 5). By contrast, rT3AM decreased the Vmax of rT3 conversion without significantly changing Km, indicating that rT3AM behaved as a noncompetitive inhibitor (Fig. 4B and Table 5). From the change in apparent Vmax for rT3, a Ki value of 1.3 ⫾ 0.2 m was calculated for rT3AM (Fig 4B and Table 5). T3AM and 3,5-T2AM, which had been identified as weak tyrosyl ring substrates of Dio1 from mouse liver membrane fractions, also acted as noncompetitive inhibitors (Table 5). As expected from the LC-MS/MS studies, which had dem-
TABLE 5. Effects of iodothyronamines on the phenolic ring deiodination of rT3 by Dio1 from mouse liver membrane fractions
Vehicle 3⬘,5⬘-T2AM (5 M) 3,3⬘-T2AM (5 M) rT3AM (5 M) T3AM (5 M) 3,5-T2AM (5 M) T4AM (5 M) 3⬘-T1AM (5 M) 3-T1AM (5 M)
Substrate of Dio1
Apparent Km (M)
Apparent Vmax (pmol/mg䡠min)
Ki (M)
Mode of inhibition
NA Phenolic ring Phenolic ring Phenolic ring Tyrosyl ring Tyrosyl ring No Dio1 substrate No Dio1 substrate No Dio1 substrate
0.43 ⫾ 0.02 1.19 ⫾ 0.1a 0.86 ⫾ 0.04a 0.42 ⫾ 0.01 0.43 ⫾ 0.03 0.42 ⫾ 0.02 0.43 ⫾ 0.03 0.42 ⫾ 0.04 0.43 ⫾ 0.03
142.7 ⫾ 2.7 136.4 ⫾ 4.8a 136.9 ⫾ 2.9a 31.3 ⫾ 2.3a 124.5 ⫾ 4.7a 134.9 ⫾ 2.1a 144.2 ⫾ 4.1 141.9 ⫾ 1.4 142.1 ⫾ 5.2
NA 2.6 ⫾ 0.4 4.8 ⫾ 1.1 1.3 ⫾ 0.2 34.0 ⫾ 2.6 85.0 ⫾ 7.7 NA NA NA
NA Mixed Mixed Noncompetitive Noncompetitive Noncompetitive NA NA NA
The apparent Km, Vmax, and Ki values are from three separate experiments performed in triplicate and are presented as mean ⫾ Not applicable. a Statistical difference with P ⬍ 0.05 as determined by Friedman test followed by Dunn`s post test.
SD.
NA,
Piehl et al. • Thyronamines Are Substrates of Deiodinases
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FIG. 4. Thyronamine substrates of Dio1 inhibit the phenolic ring deiodination of rT3 by Dio1 in 125I⫺ release assays. In Eadie-Hofstee plots (left panels), incubations with dimethylsulfoxide are designated (⽧), whereas the various thyronamine concentrations are presented as follows: 〫, 0.1 M; Œ, 0.5 M; ‚, 1.0 M; F, 2.0 M; E, 3.0 M 䡺, 5.0 M; ⫻, 7.0 M; f, 10.0 M. The effect of thyronamines on the Km and Vmax of the phenolic ring deiodination of rT3 by Dio1 was analyzed using Friedman test followed by Dunn’s post test, and P ⬍ 0.05 was considered significant. In Dixon plots (right panels), the various rT3 concentrations are presented as follows: ⽧, 0.2 M; 〫, 0.5 M; Œ, 1.0 M; ‚, 2.3 M; F, 3.3 M; E, 3.8 M; 䡺, 4.4 M. 3⬘,5⬘-T2AM acted as a mixed inhibitor (A), whereas rT3AM caused noncompetitive inhibition (B). The mean ⫾ SD values reported for apparent Km, apparent Vmax, and Ki values are from three separate experiments performed in triplicate.
onstrated weak tyrosyl ring deiodinations of T3AM and 3,5T2AM, those substrates exhibited high Ki values of 34.0 ⫾ 2.6 and 85.0 ⫾ 7.7 m, respectively (Table 5). Thyronamines, which were not converted by Dio1 from mouse liver membrane fractions in the LC-MS/MS experiments, namely T4AM, 3⬘-T1AM, and 3-T1AM, failed to show any effect on the 5⬘-deiodination of rT3 by Dio1 up to a concentration of 10 m (Table 5). Taken together, the results obtained from the competition assays were consistent with the data obtained by the LCMS/MS experiments and indicated selective deiodination reactions of the thyronamine substrates by Dio1. Discussion
The identification of thyronamines as isozyme-specific substrates of deiodinases strongly supports a role of deiodinases in the yet unknown pathways of thyronamine biosynthesis. There are two putative mechanisms of thyronamine production, none of which can be excluded yet. First of all, thyronamines might be synthesized de novo. This would require ether-bond coupling of two tyrosyl rings resembling the biosynthesis of iodothyronines in their precursor protein thyroglobulin (18). Moreover, de novo biosynthesis of 3-T1AM from T0AM would necessitate iodination of T0AM. So far, such reactions have been described only within the thyroid gland involving thyroperoxidase and dual oxidases (19). However, neither significant release of iodothyronines with lower iodination grade than T4 or T3 nor direct secretion of thyronamines from the thyroid gland has been reported yet. Second, thyronamines could be derived from thyronines by decarboxylation of the -alanine side chain. The aromatic amino acid decarboxylase has frequently been proposed as a candidate enzyme for thyronine decarboxylation partly due to its relatively broad substrate specificity including l-3,4-dihydroxyphenylalanine (l-dopa) and 5-hydroxytryptophane (20). If there were decarboxylating activities for all thyronines, e.g. if 3-T1 could be decarboxylated to 3-T1AM and T0 could be decarboxylated to T0AM, the role of
deiodinases in thyronamine biosynthesis would be an indirect one, namely synthesizing sufficient amounts of 3-T1 and T0 from T4. But if the putative decarboxylating activities were restricted to iodine-containing thyronines, i.e. 3-T1 could be decarboxylated to 3-T1AM but T0 could not be decarboxylated to T0AM, a 3-deiodinase activity would be required directly to produce T0AM from 3-T1AM (Fig. 5). With the panel of newly identified thyronamine-deiodinating reactions described here, sequential deiodination from T4AM to T0AM appears possible (Fig. 5). This panel allows confining the biosynthetic pathways for 3-T1AM and T0AM. It suggests a synthesis of endogenous T0AM from 3-T1AM via tyrosyl ring deiodination by Dio3 rather than from 3⬘-T1AM via phenolic ring deiodination. By contrast, endogenous 3-T1AM is equally likely to be synthesized from 3,3⬘-T2AM via phenolic ring deiodination and from 3,5T2AM via tyrosyl ring deiodination, provided that 3,5-T2AM or 3,3⬘-T2AM occurs in vivo. So far, only 3-T1AM and T0AM have been detected endogenously (1). The detection of thyronamines with higher iodine content in vivo might have been hampered by the fact that their mass spectrometric detection is technically more difficult. Alternatively or additionally, their levels in vivo could be very low due to putative rapid deiodination to 3-T1AM and T0AM.
FIG. 5. Summary of the newly identified thyronamine deiodination reactions.
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The identified thyronamine substrates were functionally verified by inhibiting the Dio1-catalyzed 5⬘-deiodination of rT3. In these studies, mouse liver membrane fractions were chosen as an exemplary deiodinase source because both phenolic and tyrosyl ring thyronamine substrates had been identified for this enzyme preparation in LC-MS/MS experiments. The phenolic ring substrates rT3AM, 3⬘,5⬘-T2AM and 3,3⬘-T2AM were potent inhibitors exhibiting Ki values in the lower micromolar range, i.e. comparable to the Km value of the phenolic ring deiodination of rT3. By contrast, the Ki values of the tyrosyl ring substrates were roughly one order of magnitude higher. These data were consistent with the LC-MS/MS experiments in which the phenolic and tyrosyl ring substrates were converted readily and weakly, respectively. So far, it is unclear why only some thyronamine substrates acted as mixed inhibitors with predominantly competitive elements (3⬘,5⬘-T2AM and 3,3⬘-T2AM), whereas other thyronamines unexpectedly acted as noncompetitive inhibitors (rT3AM, T3AM, and 3,5-T2AM; Table 5). Because up to now, the molecular conformation of only one thyronamine, namely T3AM, has been resolved (21), no structure-function relationships for deiodinases and thyronamines (comparable to those of deiodinases and thyronines) are available to clarify this discrepancy. However, it can be excluded that the modes of inhibition exhibit any relationship solely to the iodine substitution pattern of the thyronamine substrates or to the position of the iodine removed. In our study, we used two different Dio1 preparations, namely HepG2 lysates and mouse liver membrane fractions. Interestingly, their substrate specificities did not overlap completely. Although rT3AM and 3⬘,5⬘-T2AM were deiodinated at the phenolic rings by both preparations, 3,3⬘-T2AM, T3AM, and 3,5-T2AM were exclusively deiodinated by mouse liver membrane fractions. This difference persisted even when the pH of the homogenization buffer and the potassium phosphate buffer were increased to 8.0 (refer to Table 2, data not shown). Therefore, our data might suggest that species-specific factors modulate the Dio1 substrate specificity toward thyronamines. Besides supporting a role for deiodinases in thyronamine biosynthesis, our data also provide new insights into the structural requirements for deiodinase substrates in general. Deiodinases have long been known to convert not only iodothyronines but also sulfated iodothyronines, e.g. T4S and T3S (22), iodothyronine glucuronic acid esters, e.g. T4G (23), and iodothyronine acetic acid analogs, e.g. tetrac and triac (4) as well as N-acetylated iodothyronine derivatives (24, 25). In comparison with thyronines, these metabolites either carry additional negative charges or exhibit a negatively charged side chain. The identification of thyronamines as deiodinase substrates shows that deiodinases can indeed convert substrates with a positively charged side chain. Our analyses were based on a newly established, robust, and sensitive analytical method applying LC-MS/MS with selected reaction monitoring for thyronamine detection. This method has several advantages over classical iodide release assays. It allows analyzing deiodinations of substrates without radioactive labeling. Product formation and substrate disappearance are monitored simultaneously and are both quantifiable. Moreover, sequential
Piehl et al. • Thyronamines Are Substrates of Deiodinases
monodeiodinations are detectable. In contrast to RIAs, LC-MS/MS experiments are devoid of antibody crossreactions. Compared with radioactive assays, this LCMS/MS method is of course less sensitive and more time consuming but yields a more complete picture of deiodination cascades. Furthermore, we are not aware of any other HPLC method achieving complete baseline separation of all except one constitutional thyronamine and thyronine isomer. Thus, the LC-MS/MS method allows for backtracking the position of each iodine atom removed from the substrate, which to our knowledge has not been achieved by any other method so far. Interestingly, sequential monodeiodination cascades were observed only in the case of the tyrosyl ring deiodinations of T3 and T3AM by Dio3-containing ECC-1 lysates (Fig. 3, F and I). In all other experiments, one-step deiodinations were monitored even if the product proved to be readily convertible by the respective deiodinase when used as a direct substrate. Considering that the sensitivity of the LC-MS/MS method used here was inversely proportional to the degree of iodination of thyronamines, the predominant absence of sequential monodeiodinations indicates a substrate preference of thyronamines with higher iodine content over those with lower iodine content for deiodinases. In most instances, the thyronamine-deiodinating reactions are identical to the known deiodination reactions for the corresponding thyronines. For instance, in our study, rT3AM was readily converted by all three deiodinase isozymes, which corresponds to the deiodination reactions reported for rT3 (5). Due to the recent discovery of thyronamine sulfation by hepatic, cardiac, and brain sulfotransferases (SULTs), the spectrum of deiodinase substrates could become even broader (26). T0AM and 3-T1AM, which occur in vivo, were found to be readily sulfated by human liver SULT1A3. Moreover, 3-T1AM was sulfated by homogenates of human brain and cardiac tissue, i.e. target tissues of thyronamine action. These SULT actions might serve to attenuate and thus regulate thyronamine action. It remains to be tested whether T0AMS, 3-T1AMS, and T3AMS occur in vivo, whether they can also be deiodinated and, if so, whether thyronamine sulfation has any effect on deiodination efficiency. According to literature data on iodothyronine sulfation, Dio1 would be the most likely candidate to catalyze deiodinations of sulfated thyronamines because deiodinations of iodothyronine sulfoconjugates by Dio2 and Dio3 are very limited (4). In summary, we present here the systematic identification of thyronamines as efficient substrates for deiodinases by a novel LC-MS/MS-based method. Each deiodinase isozyme exhibited a unique substrate specificity toward thyronamines. The newly identified thyronamine substrates were functionally verified by inhibiting the 5⬘-deiodination of rT3 in a Dio1-catalyzed reaction in classical 125I⫺ release assays. These data support a role for deiodinases in thyronamine biosynthesis. Moreover, by excluding some thyronamine deiodination reactions, the biosynthetic pathways of 3-T1AM and T0AM were confined.
Piehl et al. • Thyronamines Are Substrates of Deiodinases
Acknowledgments We thank Ms. Christel Rozycki for excellent technical support. Received December 4, 2007. Accepted March 4, 2008. Address all correspondence and requests for reprints to: Prof. Dr. Josef Ko¨hrle, Institut fu¨r Experimentelle Endokrinologie und Endokrinologisches Forschungszentrum der Charite´ EnForCe´, Charite´–Universita¨tsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany. E-mail:
[email protected]. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG Graduate College 1208, TP3 to J.K.) and in part by a grant from the National Institutes of Health (DK 52798 to T.S.S.). Disclosure Statement: The authors of this manuscript have nothing to disclose.
References 1. Scanlan TS, Suchland KL, Hart ME, Chiellini G, Huang Y, Kruzich PJ, Frascarelli S, Crossley DA, Bunzow JR, Ronca-Testoni S, Lin ET, Hatton D, Zucchi R, Grandy DK 2004 3-Iodothyronamine is an endogenous and rapidacting derivative of thyroid hormone. Nat Med 10:638 – 642 2. Chiellini G, Frascarelli S, Ghelardoni S, Carnicelli V, Tobias SC, Debarber A, Brogioni S, Ronca-Testoni S, Cerbai E, Grandy DK, Scanlan TS, Zucchi R 2007 Cardiac effects of 3-iodothyronamine: a new aminergic system modulating cardiac function. FASEB J 21:1597–1608 3. Weatherman RV 2007 A triple play for thyroid hormone. ACS Chem Biol 2:377–379 4. Wu SY, Green WL, Huang WS, Hays MT, Chopra IJ 2005 Alternate pathways of thyroid hormone metabolism. Thyroid 15:943–958 5. Kohrle J 2002 Iodothyronine deiodinases. Methods Enzymol 347:125–167 6. Hart ME, Suchland KL, Miyakawa M, Bunzow JR, Grandy DK, Scanlan TS 2006 Trace amine-associated receptor agonists: synthesis and evaluation of thyronamines and related analogues. J Med Chem 49:1101–1112 7. Miyakawa M, Scanlan TS 2006 Synthesis of [125I]-, [2H]-, and [3H]-labeled 3-iodothyronamine (T1AM). Synth Comm 36:891–902 8. Kohrle J 2005 Selenium and the control of thyroid hormone metabolism. Thyroid 15:841– 853 9. Shah VP, Midha KK, Findlay JW, Hill HM, Hulse JD, McGilveray IJ, McKay G, Miller KJ, Patnaik RN, Powell ML, Tonelli A, Viswanathan CT, Yacobi A 2000 Bioanalytical method validation; a revisit with a decade of progress. Pharm Res 17:1551–1557 10. Peters FT, Drummer OH, Musshoff F 2007 Validation of new methods. Forensic Sci Int 165:216 –224
Endocrinology, June 2008, 149(6):3037–3045
3045
11. Schmutzler C, Hamann I, Hofmann PJ, Kovacs G, Stemmler L, Mentrup B, Schomburg L, Ambrugger P, Gruters A, Seidlova-Wuttke D, Jarry H, Wuttke W, Kohrle J 2004 Endocrine active compounds affect thyrotropin and thyroid hormone levels in serum as well as endpoints of thyroid hormone action in liver, heart and kidney. Toxicology 205:95–102 12. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248 –254 13. Kester MH, Kuiper GG, Versteeg R, Visser TJ 2006 Regulation of type III iodothyronine deiodinase expression in human cell lines. Endocrinology 147: 5845–5854 14. Leonard JL, Rosenberg IN 1980 Iodothyronine 5⬘-deiodinase from rat kidney: substrate specificity and the 5⬘-deiodination of reverse triiodothyronine. Endocrinology 107:1376 –1383 15. Jakobs TC, Mentrup B, Schmutzler C, Dreher I, Kohrle J 2002 Proinflammatory cytokines inhibit the expression and function of human type I 5⬘deiodinase in HepG2 hepatocarcinoma cells. Eur J Endocrinol 146:559 –566 16. Curcio C, Baqui MM, Salvatore D, Rihn BH, Mohr S, Harney JW, Larsen PR, Bianco AC 2001 The human type 2 iodothyronine deiodinase is a selenoprotein highly expressed in a mesothelioma cell line. J Biol Chem 276:30183–30187 17. Schoenmakers CH, Pigmans IG, Visser TJ 1992 Species differences in liver type I iodothyronine deiodinase. Biochim Biophys Acta 1121:160 –166 18. Dunn JT, Dunn AD 2001 Update on intrathyroidal iodine metabolism. Thyroid 11:407– 414 19. Ris-Stalpers C 2006 Physiology and pathophysiology of the DUOXes. Antioxid Redox Signal 8:1563–1572 20. Zhu MY, Juorio AV 1995 Aromatic l-amino acid decarboxylase: biological characterization and functional role. Gen Pharmacol 26:681– 696 21. Cody V, Meyer T, Dohler KD, Hesch RD, Rokos H, Marko M 1984 Molecular structure and biochemical activity of 3,5,3⬘-triiodothyronamine. Endocr Res 10:91–99 22. Visser TJ 1994 Role of sulfation in thyroid hormone metabolism. Chem Biol Interact 92:293–303 23. Hays MT, Cavalieri RR 1992 Deiodination and deconjugation of the glucuronide conjugates of the thyroid hormones by rat liver and brain microsomes. Metabolism 41:494 – 497 24. Kohrle J, Hesch RD 1984 Biochemical characteristics of iodothyronine monodeiodination by rat liver microsomes: the interaction between iodothyronine substrate analogs and the ligand binding site of the iodothyronine deiodinase resembles that of the TBPA-iodothyronine ligand binding. Horm Metab Res Suppl 14:42–55 25. Koehrle J, Auf’mkolk M, Rokos H, Hesch RD, Cody V 1986 Rat liver iodothyronine monodeiodinase. Evaluation of the iodothyronine ligand-binding site. J Biol Chem 261:11613–11622 26. Pietsch CA, Scanlan TS, Anderson RJ 2007 Thyronamines are substrates for human liver sulfotransferases. Endocrinology 148:1921–1927
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