J. Endocrinol. Invest. 13: 901-904, 1990
Dyshormonogenetic goiter: presence of an inhibitor of normal human thyroid peroxidase1 D. Rosenthal*, D. P. Carvalho-Guimaräes*, M. Knobel**, and G. A. Medeiros-Neto** *Laborat6rio de Fisiologia End6crina, Institute de Bioffsica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21949, Rio de Janeiro, and **Laborat6rio de Tire6ide, Hospital das Clfnicas, Universidade de Säo Paulo, 05403 Säo Paulo, Brasil
ABSTRACT. A dialyzable, thermostable inhibitor of normal thyroid peroxidase (TPO), with UV absorption maximum at 250-260 nm, was found in the digitonized, washed particulate fraction of two dyshormonogenetic goiters. No intrinsic TPO iodide-oxidation activity was detectable in either of these goiters, and their TPO iodination activity
was below the method sensitivity threshold, even after dialysis. These findings could be explained by an absent or abnormal TPO associated with the synthesis of a TPO-inhibitor, or by the irreversible inhibition of a normal enzyme by the inhibitor.
INTRODUCTION Thyroid peroxidase (TPO) can be shown to mediate at least 3 different reactions in vitra. iodide oxidation, protein iodination or iodine organification, and coupling of iodotyrosines (1-3). In vivo, a diminished TPO activity is suggested by a significant radioiodide discharge from the thyroid gland after administration of perchlorate or thiocyanate. The degree of TPO activity impairment can be slight, as reported in some cases of Hashimoto's thyroiditis (4), or it can be marked, as in some types of dyshormonogenetic goiter or goitrous hypothyroidism (5). The defect may be due to a qualitative or quantitative impairment of the enzyme or of its protein substrate. Other abnormalities, such as the presence of a TPO inhibitor, must also be considered.
DG2), a diffuse toxic goiter (DTG) and the normal paranodular portion of a nodular goiter (PN) were obtained at thyroidectomy, immediately frozen and stored at -20 C, The dyshormonogenetic goiter patients were not related but were from families with a high incidence of goiter (with 2 goitrous siblings each). Both had positive perchlorate discharge tests (Ioss of more than 40% of thyroid radioiodide 2h after perchlorate administration) and were said to have goiter at birth. One patient (DG1 ) had been hypothyroid since birth, a previous thyroidectomy being followed by goiter regrowth, While off thyroid medication his serum T 4 was 1.0-2.0 ~g/dl. serum T3 40-70ng/dl and serum TSH 45-146~U / ml; thyroid 131-1 uptake was 17% at 2h and 4% at 24h, The other patient (DG2) had low serum T4 (2~g/dl), normal serum T3 (143ng/dl) and an exaggerated serum TSH response to exogenous TRH (basal: 6.4~U/ml; peak value: 102~U/ml), but was not clinically hypothyroid. The thyroid 131-1 uptake was increased (70% at 2h and 55% at 24h). Peroxidase was extracted as previously described (6,7). In short, the thyroid tissue sampies were cleaned, homogenized in 50mM TrisHCI buffer, pH 7.2, containing 1 mM KI, and centrifuged (27,000 xg, 4 C, 120 min). The pellets were washed once, then suspended in 2 ml digitonin (1 % w Iv) and incubated· at 4 C for 24h, The digitonin-
MATERIALS AND METHODS Sam pies from two dyshormonogenetic goiters (DG1,
'Research supported in part by FINEP. FAPERJ and CNPq.
Key·words:Thyroid gland. dyshormonogenetic goiter. thyroid peroxidase. peroxidase inhibitor. hypothyroidism.
Correspondence: Dr. D. Rosenthai. Instituto de Biofisica Carlos Chagas. Filho. UFRJ, Centro de Ci~ncias da Saude, Bloco G, 21949, Rio de Janeiro, RJ, Brasil. Received May 31, 1990; accepted September 6, 1990.
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porated/mg TPQ protein). In addition to minimal or nondetectable activity, both OG preparations contained TPQ inhibitors. The OG1 preparation inhibited the OTG-TPQ iodine organification aetivity (Fig. 1), but the inhibition of iodine organification activity by OG2 was not tested due to insufficient material. Both OG1 (data not shown) and OG2 preparations inhibited the OTG-TPQ iodide-oxidation aetivity in a dose-dependent manner, while an additive effect was produeed by PN-TPQ (Fig. 2). The TPQ-inhibitory activity was not affected by heating (100 C for 30 min) but was markedly decreased by dialysis (18h, 4 C) against homogenizing buffer (Fig. 2). No intrinsie iodide-oxidation activity eould be detected in the dialized OG2-TPQ preparation, although the 18h dialysis procedure did not signifieantly affeet the normal PN-TPQ iodide-oxidation activity (89% of the original aetivity preserved). The UV absorption speetra of the OG1 and OG2 preparations differed from those of diffuse toxie goiter and paranodular tissue TPQ preparations by markedly increased absorption between 250 and 260nm. Most of this difference disappeared after dialysis (Fig. 3).
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Fig.1 - Thyroid peroxidase (TPO) iodide organification inhibition by dyshormonogenetic goiter TPO preparation. Digitonin-treated particulate fractions of homogenates from diffuse toxic goiter (DTG-TPO) were incubated with 30nmoles 125 1_, 660j.Jg bovine serum albumin as iodide acceptor and glucoseglucose oxidase as the H2 0 2 generating system (pH 7.4, 37 C, 30min). Iodide organification by the DTG- TPO preparation was asseyed without (hatched column) and with (empty column) different amounts of the DG.l TPO preparation.
treated suspensions were centrifuged (27,000 g, 4 C, 90 min), and the supernatants, containing solubilized TPQ, were used for the activity and inhibitory assays. The protein concentration of the solubilized TPQ preparations was determined by the method of Warburg and Christian (8). The TPQ activity was determined by the iodide-oxidation assay as previously reported (7), and by an iodide organification assay based on the methods of Nakashima and Taurog (9) and Nagataki et al. (10), using 660 ug bovine serum albumin (BSA) as iodine acceptor and 30 min incubation at 37 C (11 ). An active (OTG) TPQ was assayed in the presence of different amounts of the OG TPQ preparations in order to estimate their TPQ-inhibitory activity. Results are given as percentage of the original OTGTPQ activity.
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Fig. 2 - Thyroid peroxidase (TPO) iodide oxidation inhibition by dyshormonogenetic goiter TPO preparation. Oigitonin-treated particulate fractions from diffuse tOXIC goiter (OTG- TPO), dyshormonogenetic goiter (DG.2) and paranodular thyroid tissue (PN) were used for assays. The iodide-oxidation activity of OTG- TPO was assayed (40fJg enzyme, 24mM KI, pH 74 and glucose-glucose oxidase as the H2 0 2 generating system) without (0) and with different amounts of the following preparations.' 1. PN, 2. OG.2; 3. OG.2 exposed to 100 C tor 30 min, 4 and 5. OG.2 dialyzed against homogenizing buffer (4 C) for Bh or IBh, respectively Results are given as percentage of the original OTG- TPO activity
RESULTS The iodide-oxidation activities of the OTG (3865 U/g protein) and PN (895 U/g protein) sampies are within our previously determined ranges (10), while no activity was detected in either of the dyshormonogenetic goiters. The iodide-organification activity was also negligible (nondetectable in OG1, and below the sensitivity threshold for DG2) as compared to the activity from DTG (109.4 nmol I-incor-
902
TPO inhibitor in dyshormonogenetic goiter
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absorbance peak and the DTG-TPO-inhibitory activity (Figs. 2, 3). This could be due to an absent or abnormal TPO or to an irreversible inhibition of a normal enzyme by the inhibitor. At the moment we have no data indicating which of these possibilities could be true. A genetic error in the TPO molecule, resulting in a TPO inhibitor, is one possible way to explain our results. Other possibilities such as the inappropriate increase of anormal TPO-activity regulator, supported by the identificaticin of a TPO-inhibitor in nondyshormonogenetic goiters (13) and normal animal thyroid glands (15,16), must also be considered. Further studies are needed to better characterize this TPO-inhibitory substance and establish its physiological significance, if any.
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Dyshormonogenetic goiter: presence of an inhibitor of normal human thyroid peroxidase1 D. Rosenthal*, D. P. Carvalho-Guimaräes*, M. Knobel**, and G. A. Medeiros-Neto** *Laborat6rio de Fisiologia End6crina, Institute de Bioffsica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21949, Rio de Janeiro, and **Laborat6rio de Tire6ide, Hospital das Clfnicas, Universidade de Säo Paulo, 05403 Säo Paulo, Brasil
ABSTRACT. A dialyzable, thermostable inhibitor of normal thyroid peroxidase (TPO), with UV absorption maximum at 250-260 nm, was found in the digitonized, washed particulate fraction of two dyshormonogenetic goiters. No intrinsic TPO iodide-oxidation activity was detectable in either of these goiters, and their TPO iodination activity
was below the method sensitivity threshold, even after dialysis. These findings could be explained by an absent or abnormal TPO associated with the synthesis of a TPO-inhibitor, or by the irreversible inhibition of a normal enzyme by the inhibitor.
INTRODUCTION Thyroid peroxidase (TPO) can be shown to mediate at least 3 different reactions in vitra. iodide oxidation, protein iodination or iodine organification, and coupling of iodotyrosines (1-3). In vivo, a diminished TPO activity is suggested by a significant radioiodide discharge from the thyroid gland after administration of perchlorate or thiocyanate. The degree of TPO activity impairment can be slight, as reported in some cases of Hashimoto's thyroiditis (4), or it can be marked, as in some types of dyshormonogenetic goiter or goitrous hypothyroidism (5). The defect may be due to a qualitative or quantitative impairment of the enzyme or of its protein substrate. Other abnormalities, such as the presence of a TPO inhibitor, must also be considered.
DG2), a diffuse toxic goiter (DTG) and the normal paranodular portion of a nodular goiter (PN) were obtained at thyroidectomy, immediately frozen and stored at -20 C, The dyshormonogenetic goiter patients were not related but were from families with a high incidence of goiter (with 2 goitrous siblings each). Both had positive perchlorate discharge tests (Ioss of more than 40% of thyroid radioiodide 2h after perchlorate administration) and were said to have goiter at birth. One patient (DG1 ) had been hypothyroid since birth, a previous thyroidectomy being followed by goiter regrowth, While off thyroid medication his serum T 4 was 1.0-2.0 ~g/dl. serum T3 40-70ng/dl and serum TSH 45-146~U / ml; thyroid 131-1 uptake was 17% at 2h and 4% at 24h, The other patient (DG2) had low serum T4 (2~g/dl), normal serum T3 (143ng/dl) and an exaggerated serum TSH response to exogenous TRH (basal: 6.4~U/ml; peak value: 102~U/ml), but was not clinically hypothyroid. The thyroid 131-1 uptake was increased (70% at 2h and 55% at 24h). Peroxidase was extracted as previously described (6,7). In short, the thyroid tissue sampies were cleaned, homogenized in 50mM TrisHCI buffer, pH 7.2, containing 1 mM KI, and centrifuged (27,000 xg, 4 C, 120 min). The pellets were washed once, then suspended in 2 ml digitonin (1 % w Iv) and incubated· at 4 C for 24h, The digitonin-
MATERIALS AND METHODS Sam pies from two dyshormonogenetic goiters (DG1,
'Research supported in part by FINEP. FAPERJ and CNPq.
Key·words:Thyroid gland. dyshormonogenetic goiter. thyroid peroxidase. peroxidase inhibitor. hypothyroidism.
Correspondence: Dr. D. Rosenthai. Instituto de Biofisica Carlos Chagas. Filho. UFRJ, Centro de Ci~ncias da Saude, Bloco G, 21949, Rio de Janeiro, RJ, Brasil. Received May 31, 1990; accepted September 6, 1990.
901
D. Rosenthai, D. P. Carvalho-Guimaraes, M. Knobel, et al.
~
30
(J)
porated/mg TPQ protein). In addition to minimal or nondetectable activity, both OG preparations contained TPQ inhibitors. The OG1 preparation inhibited the OTG-TPQ iodine organification aetivity (Fig. 1), but the inhibition of iodine organification activity by OG2 was not tested due to insufficient material. Both OG1 (data not shown) and OG2 preparations inhibited the OTG-TPQ iodide-oxidation aetivity in a dose-dependent manner, while an additive effect was produeed by PN-TPQ (Fig. 2). The TPQ-inhibitory activity was not affected by heating (100 C for 30 min) but was markedly decreased by dialysis (18h, 4 C) against homogenizing buffer (Fig. 2). No intrinsie iodide-oxidation activity eould be detected in the dialized OG2-TPQ preparation, although the 18h dialysis procedure did not signifieantly affeet the normal PN-TPQ iodide-oxidation activity (89% of the original aetivity preserved). The UV absorption speetra of the OG1 and OG2 preparations differed from those of diffuse toxie goiter and paranodular tissue TPQ preparations by markedly increased absorption between 250 and 260nm. Most of this difference disappeared after dialysis (Fig. 3).
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Fig.1 - Thyroid peroxidase (TPO) iodide organification inhibition by dyshormonogenetic goiter TPO preparation. Digitonin-treated particulate fractions of homogenates from diffuse toxic goiter (DTG-TPO) were incubated with 30nmoles 125 1_, 660j.Jg bovine serum albumin as iodide acceptor and glucoseglucose oxidase as the H2 0 2 generating system (pH 7.4, 37 C, 30min). Iodide organification by the DTG- TPO preparation was asseyed without (hatched column) and with (empty column) different amounts of the DG.l TPO preparation.
treated suspensions were centrifuged (27,000 g, 4 C, 90 min), and the supernatants, containing solubilized TPQ, were used for the activity and inhibitory assays. The protein concentration of the solubilized TPQ preparations was determined by the method of Warburg and Christian (8). The TPQ activity was determined by the iodide-oxidation assay as previously reported (7), and by an iodide organification assay based on the methods of Nakashima and Taurog (9) and Nagataki et al. (10), using 660 ug bovine serum albumin (BSA) as iodine acceptor and 30 min incubation at 37 C (11 ). An active (OTG) TPQ was assayed in the presence of different amounts of the OG TPQ preparations in order to estimate their TPQ-inhibitory activity. Results are given as percentage of the original OTGTPQ activity.
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Fig. 2 - Thyroid peroxidase (TPO) iodide oxidation inhibition by dyshormonogenetic goiter TPO preparation. Oigitonin-treated particulate fractions from diffuse tOXIC goiter (OTG- TPO), dyshormonogenetic goiter (DG.2) and paranodular thyroid tissue (PN) were used for assays. The iodide-oxidation activity of OTG- TPO was assayed (40fJg enzyme, 24mM KI, pH 74 and glucose-glucose oxidase as the H2 0 2 generating system) without (0) and with different amounts of the following preparations.' 1. PN, 2. OG.2; 3. OG.2 exposed to 100 C tor 30 min, 4 and 5. OG.2 dialyzed against homogenizing buffer (4 C) for Bh or IBh, respectively Results are given as percentage of the original OTG- TPO activity
RESULTS The iodide-oxidation activities of the OTG (3865 U/g protein) and PN (895 U/g protein) sampies are within our previously determined ranges (10), while no activity was detected in either of the dyshormonogenetic goiters. The iodide-organification activity was also negligible (nondetectable in OG1, and below the sensitivity threshold for DG2) as compared to the activity from DTG (109.4 nmol I-incor-
902
TPO inhibitor in dyshormonogenetic goiter
2.0 - , - - - - - - - - - - - - - - - - - - ,
absorbance peak and the DTG-TPO-inhibitory activity (Figs. 2, 3). This could be due to an absent or abnormal TPO or to an irreversible inhibition of a normal enzyme by the inhibitor. At the moment we have no data indicating which of these possibilities could be true. A genetic error in the TPO molecule, resulting in a TPO inhibitor, is one possible way to explain our results. Other possibilities such as the inappropriate increase of anormal TPO-activity regulator, supported by the identificaticin of a TPO-inhibitor in nondyshormonogenetic goiters (13) and normal animal thyroid glands (15,16), must also be considered. Further studies are needed to better characterize this TPO-inhibitory substance and establish its physiological significance, if any.
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