Triiodothyronine Sulfate Metabolism in Euthyroid Man

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in euthyroid man, whereas alternate pathways, including sulfate and glucuronide conjugation, conversion to acetic acid analogs and ether-link cleavage ...
0021-972X/91/7304-0703$02.00/0 0021-972X/91/7~04-07oa$02.00/0 .Journal Journal of Clinical Endocrinology and Metabolism (CI 1991 Copyright (ci 1991 by The Endocrine Society

No. 4 Vol. 73, No.4 U.S.A. Printed in U.S.A.

Characteristics of 3,5,3' -Triiodothyronine Sulfate 3,5,3'-Triiodothyronine Metabolism in Euthyroid Man* JONATHAN S. LoPRESTI, LYNN MIZUNO, ANANDA NIMALYSURIA, KENNETH P. ANDERSON, CAROLE A. SPENCER, AND JOHN T. NICOLOFF Uniuersity University of Southern California, California, School SchoolofofMedicine, Medicine,Department DepartmentofofMedicine, Medicine,2025 2025Zonal ZonalAuenue, Avenue, Los Angeles, Angeles, California California90033 90033

< 0.003). 0.003). However, no no relationship between clearance and deiod· deiod-

ABSTRACT. The sulfated sulfated conjugate of T 33 (T33S) S) has long been recognized as a normal product of peripheral thyroid hormone S may metabolism. In order to better understand the role that T33S play in this process, the metabolic handling of T33S S was studied in euthyroid man. After After the iv administration of [l25I]T [125I]T33SS in S was found to be rapidly metabolized with estimated man, T33S after a bolus injection mean MCR of 135 ± 15 liters/day (LID) (L/D) after and 127 ± 8 L/D employing a constant infusion. The primary of route of T33S S disposal was by deiodination with an efficiency efficiency of 92%. The administration of propylthiouracil (PTU, 300 mg every every 6hx X 55days) days) and and iopanoic iopanoic acid acid (lA, (IA,500 500mg mgevery everyday day xx 55days), days), both inhibitors of deiodination, decreased clearance compared LID, P < 0.01 and 46 ± 10 L/D, LID, P < 0.002, to control (87 ± 9 L/D, T3S respectively). A 3-day fast also reduced the clearance of T 3S (56 ± 10 L/D, P < 0.002). All three maneuvers decreased the total 91 ± 2%, urinary deiodination fraction of tracer T33S (control 91 PTU 70 ± 9%, P < 0.04, IA 26 ± 3%, 0.0001, and fasting 3%, P < 0.0001, 58 ± 6%, P < 0.01). A strong correlation between T T3S 3S clearance and deiodination was noted for fasting and IA only (r = 0.78, P

ination was noted with PTU administration presumably as a result of a compensatory increase in biliary losses of T 33S, S. The urinary thyronine excretion pattern demonstrated the presence 3,3'-T of small amounts of labeled T33,3,3'-T -T22SS with the ,3,3' ·T22, and 3,3' major metabolite being T3S itself. TSH levels were not influS designed to achieve a serum infusion of stable T33S enced by the infusion value greater than 50 ng/dL. No absorption of intact T33S was detected after after its oral ingestion. In conclusion, T33S S is rapidly cleared from the serum, primarily by deiodination, may undergo nondeiodinative disposal when hepatic deiodination is inhibited by PTU but not with IA or fasting, and has no intrinsic biological activity. Thus, T33S may serve as a metabolite of T Ta3 for its rapid deiodinative disposal. Although the precise role T33S S plays in human thyroid hormone metabolism has not been defined, the metabolic characteristics of T TaS 3S appear similar to that of an unidentified unidentified alternate T44 metabolite formed in low T 33 states of of fasting and nonthyroidal illness. (J (J Clin Clin Endocrinol Metab 73: 73: Endocrinol Metab 703-709,1991) 703-709, 1991)

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as yet unidentified unidentified alternate disposal pathway must occur (7). The question then arises as to which alternate pathway is responsible for this apparent "accounting gap" in T 44 disposal. Hypertrophy of a number of deiodinative and nondeiodinative pathways could theoretically occur in low T 33 states which would account for this gap in disposal. However, as recent studies from our laboratory have shown that the overall T44 deiodination rate remains constant in fasting, the alternate metabolite formed must serve as an excellent substrate for subsequent deiodination (8). Further, its production must be reciprocally increased as T 33 generation from T44 falls, thus allowing T44 disposal and deiodination rates to remain unaltered T33 sulfate (T33S) appears to (9). Based on these criteria, T unidentified metabbe a suitable candidate for such an unidentified olite. In earlier studies, T33S S has been demonstrated to be a major product of T 33 metabolism in both normal and hepatectomized dogs, indicating that the sulfation sulfation of T 33 can occur both intra- and extrahepatically (10). More recently, it has been demonstrated that T3S T3S is generated

| » U URRENT R R E N T evidence indicates that the principle V y route for T 44 disposal in man occurs by either inner or outer ring deiodination forming rT 33 and T33,, respectively (1). It is estimated that the combined production of T 33 and rT 33 accounts for 80% of total T44 metabolism in euthyroid man, whereas alternate pathways, including sulfate and glucuronide conjugation, conversion to acetic acid analogs and ether-link cleavage products, are responsible for the remaining 20% (2-4). However, this pattern of T44 disposal may be substantially altered in the "low T 33 state" of fasting and nonthyroidal illness (NT!) (NTI) (5, 6). In the low T T33 state, less T T33 is generated from T4,, whereas rT rT33 production appears to remain constant. As the sum of T 33 and rT 33 production is reduced, and total T4 production and disposal is unaltered, hypertrophy of 31, 1990. Received July 31,1990. Address requests for reprints to: Jonathan S. LoPresti, M.D., Ph.D., University of Southern California, School of Medicine, 2025 Zonal Avenue, Los Angeles, California California 90033. •* This study was was supported by Grants DK-11727 and General Clinical Research Centers Program Grant M01-RR-43 from the National Institutes of Health.

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LOPRESTI ET AL. AL.

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during the metabolism of T 3a in both euthyroid man and rat (11-13). Indeed, Engler et al. al. (14) have speculated that the 50% 50% gap in T 3a disposal in euthyroid man may T3S production. Finally, in a series be accounted for by TaS al. (13) and Otten et al. of recent studies by Eedkman et al. al. preferred substrate for (15), T 3S has been shown to be a preferred 5'-deiodinase (5'-D) the hepatic type I 5' -deiodinase (5' -D) enzyme systems both in vitro and in vivo. vivo. The present investigation was undertaken to evaluate the metabolic handling of T 3 S in euthyroid man. This S metabolic clearance and involved the estimation of T 33S efficiency in a basal state as well as during deiodination efficiency the inhibition of deiodination produced by propylthiouracil (PTU), iopanoic acid (lA), (IA), and fasting (16, (16, 17, 17, 18). Estimates of oral absorption and in vivo vivo biological activity and in vitro protein binding were also evaluated. The results of these studies demonstrated that T 3a8 S is rapidly cleared from serum, primarily by deiodination, is poorly absorbed from the gastrointestinal tract, and has no detectable biological activity. Thus, T TaS 3 S would appear to possess many of the features of the missing metabolite in the low T 3a state of fasting and NT!. NTI.

as

Materials and Methods Nine healthy, euthyroid male subjects, ages 20-48 yr, participated in this study. A screening history and and physical examination were normal. No biochemical evidence of either hepatic or renal disease were present. None of the subjects had recently taken any any medications known to interfere with thyroid funcspecifically administered as as part of the the protocol. tion, unless specifically was obtained from each volunt~~er volunteer Written informed consent was the initiation of the protocol. The The project was reviewed before the by the the Institutional Review Board of the the Los and approved by Los Angeles County/University of Southern California California Medical (IRB 02976) and and conducted on on the the General Clinical Center ORB All patients were adminResearch Center of that institution. All drops of of aa supersaturated supersaturated solution solution of of potassium potassium iodide iodide istered 10 drops day before before and and daily daily throughout throughout the the study study to to inhibit inhibit thyroidal thyroidal 1 day and recycling of radio radioiodide. uptake and iodide.

Protocol design Protocol design 125 The kinetic characteristics of [125 I]T by I]Ta3SS were determined by both a pulse injection and and constant infusion. Seven volunteers 25 I]T received a bolus iv iv injection of P [125 I]Ta3S. A 21-gauge heparin lock was placed in a peripheral arm vein and maintained patent with a heparin solution (25 (25 JL/mL). /z/mL). At 1000 1000 h, h, an iv iv bolus 25I]T injection of 25 JLCi ^Ci [1 [125 I]Ta3SS in a 5% human albumin solution was administered after after having been passed through a 0.22 0.22 JLM /zM Millipore filter (Millipore Corp., Bedford, MA) to ensure stesterility. Three of the patients had blood samples obtained at time o0 and and at 2, 2, 4, 4, 6, 6, 8, 8, 10, 10, 20, 20, 30, 30, 60, 60, 90, 90, and and 120 min min to to determine the serum disappearance pattern of tracer T 3as. S. The The remaining four subjects had had additional blood samples drawn at 150, 180, 180, 240, 300, 360, 480, and and 600 600 min. min. Three of these subjects also 25I] 25I]T received 50 1] 50 JLCi itCi [1 [125 I]Tas by a constant iv iv infusion. The The [[1125 3S by

JCE&M JCE & Mo •1991 1991 Vol 73 o No4 Vol73«No4

T 33S was was mixed with a 5% 5% human serum albumin solution in 500 cc cc normal saline and and delivered at 100 100 cc/h cc/h for for 5 h via via a multispeed transmission Harvard pump (model 901). 901). Blood samples were obtained every 30 30 min min during the the infusion infusion to determine the TaS. the MCR of tracer T 3 S. Four patients also participated in studies to determine the influence of inhibition of type I 5'-D 5'-D activity on on the the serum 125 S. These inhibitory agents or disappearance pattern of [125I]T [ I]Ta3 maneuvers included administration of PTU, lA, and PTU, IA, and fasting fasting (16, (16, 17, 17, 18). 18). Each Each subject subject underwent underwent four four consecutive consecutive studies studies during during aa 3-week 3-week period. period. After After the the completion completion of of the the baseline baseline study, study, the the subjects subjects were were sequentially sequentially given given PTU PTU (300 (300 mg mg orally orally IA (500 mg load, then 500 mg daily every 6 h for 5 days), every 6 h for 5 days), IA (500 mg load, then 500 mg daily for for 55 days), and a 5-day fast. On the third day of each period the days), and a 5-day fast. On the third day of each period the 125 [125I]T S kinetic study was performed. A minimum of 5 days 3 [ I]T3S kinetic study was performed. A minimum of 5 days was emwas allowed allowed between between each each tracer tracer injection. injection. In In the the studies studies employing PTU, lA, and fasting, additional samples were obtained ploying PTU, IA, and fasting, additional samples were obtained at 720, 840, 1200, 1440 min. Blood samples from all kinetic at 720, 840, 1200, 1440 min. Blood samples from all kinetic studies were allowed to clot for 2 h at room temperature before studies were allowed to clot for 2 h at room temperature before the serum was separated and stored at -20 C until processed. the serum was separated and stored at25 —20 C until processed. was A dual isotopic technique using [1125I]TaS and [1alI]Na A dual isotopic technique using [ I]T3S and [131I]Na was employed to estimate the deiodination rate of T as with the employed to estimate the deiodination rate of T S with the I]Na acting as an internal standard to correct for3 variations [131 [131I]Na acting as an internal standard to correct for variations in the renal handling of iodide (19). After an oral water load, in the renal handling of iodide (19). After an oral water load, 251] four subjects were simultaneously administered 20 J,LCi [1125 four subjects were simultaneously administered 20 /zCi [ I] T 3S and 20 J,LCi [131I]Na iv with the urine being collected over T3S and 20 ttCi [131I]Na iv with the urine being collected over 1251] the next 3 days to measure the cumulative excretion of [125 the next 3 days to measure the cumulative excretion of [ I] 131 and [1311]. In two of these subjects, the study was repeated after and [ I]. In two of these subjects, the study was repeated after the administration of PTU (200 mg every hour for 6 h, starting the administration of PTU (200 mg every hour for 6 h, starting 2 h before the injection). In other studies, urine samples were 2 h before the injection). In other studies, urine samples were collected in 2 L plastic bottles containing 2 mL 25% human collected in 2 L plastic bottles containing 2 mL 25% human serum albumin (Travenol Laboratories, Inc., Glendale, CA) to serum albumin (Travenol Laboratories, Inc., Glendale, CA) to bind and stabilize the excreted thyronine conjugates. These bind and stabilize the excreted thyronine conjugates. These timed timed urine urine samples samples were were collected collected to to estimate estimate total total urinary urinary tracer T as deiodination and the urinary thyronine tracer T3S deiodination and the urinary thyronine conjugate conjugate the patterns. patterns. All All urine urine samples samples were were stored stored at at 44 C C during during the collection period and remained refrigerated until processed. collection period and remained refrigerated until processed. Greater Greater than than 95% 95% of of the the injected injected radioactive radioactive tracer tracer was was acaccounted for by the serial urine collections. counted for by the serial urine collections.25 To To determine determine the the125oral oral absorption absorption of of P [125I]T I]Ta3S, S, two two subjects subjects 125I]Tas orally mixed in 20 mL 1% human 25 JLCi [ were given were given 25 /uCi [ I]T3S orally mixed in 20 mL 1% human albumin water. Blood were drawn albumin solution solution in in distilled distilled water. Blood samples samples were drawn every every 30 30 min min for for 66 hh and and concurrent concurrent urine urine samples samples were were collected h. collected for for 72 72 h. To To assess assess any any potential potential biological biological activity activity of of TaS, T3S, two two subsubjects underwent the the following 6-day protocol. Each patient received a 2-day control infusion was infusion of normal saline. This was followed by a 48-h infusion infusion of [125I]T [125I]Ta3SS in inaa 5% 5% human human albumin albumin solution in saline calculated to deliver an estimated serum concentration of 50 ng/DL TaS T3S and and to complete the the protocol, an infusion infusion of normal saline was administered the remaining 2 days. Serial serum samples were drawn at 1000, 1000, 1400, 1400, 1800, 1800, and 0600 h daily to determine serum T a3, T 44,, and and 2200, 0200, and TSH values during the 6-day study. In vitro studies were performed performed to determine if T3S binds to 25 I]T [125 I]Ta3SS was treated serum proteins. Serum equilibrated with [1 with an equal volume of 10% trichloroacetic acid to precipitate (n = = 8). The mixture was centrifuged centrifuged for for 30 30 the serum proteins (n

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CHARACTERISTICS OF T 33S METABOLISM IN EUTHYROID MAN min at 3000 rpm, the supernatant decanted, and the precipitate counted. Quantification S in serum Quantification of the free fraction of T 33S was determined by equilibrium dialysis of [1251]T3S [125I]T3S labeled serum against phosphate buffer. Dialysis of serum labeled with 125 251]T [1 1]T [125 I]T33 and [[125 I]T44 was was also also performed for for comparison (n (n = = 6). These studies were carried out by Nichols Institute Reference Laboratories (San Juan Capistrano, CA). To concentrate the [1251]T [l25I]T33SS from from serum serum and and separate separate itit from from radioiodine and iodoproteins, a previously described methodology using C-18 Sep-Pak cartridges (Waters & Associates, Milford, MA) was employed (17). (17). The eluted radiothyronine fractions were collected and counted on a dual channel 'Y-counter 7-counter (TM Analytic, Elk Grove Village, IL). After After appropriate corrections for recovery, serum tracer T 33S S disappearance curves were constructed and analyzed by noncompartmental means to estimate MCR (20). (20). The MCR estimate, during the 251]T constant infusion infusion of [1 [125 I]T33S, S, was calculated by dividing the infusion infusion rate by the final plasma [1251]T [125I]T33SS concentration at equilibrium. Selected urine samples were acidified to a pH of of N HCI HCl and and processed processed as as above above to to isolate isolate the the labeled labeled 3.5 with 1 N thyronines. The urinary iodothyronine fractions were then characterized by high pressure liquid chromatography (HPLC) using a reverse phase C18 column run with a previously de26% tetrahydrofutetrahydrofuscribed gradient system ranging from 16 to 26% 125 251]T (16). Standards of [125 I]T3, [1[125 I]T33S, 1]T ran:ammonium acetate (16). 125 125 25 125 251]3,3' [[1125 I]3,3'-T , [ I]3,3'-T S, and [ I]Na were run for compari-T2' [ 1]Na were compari2 [1 1]3,3' -T 2 2 son. A modification modification of the method of Mol and Visser (21) using chlorosulfonic acid and iV,iV-dimethylformamide N,N-dimethylformamide was employed 251]T [126 I]T3S to synthesize the [1 3S used in the present studies. Instead of allowing the reaction to proceed overnight at room temper251]T ature, the generation of [1[125 I]T3SS was carried out at 37 C for 2 25 IJT 25 IJT [125 I]T33SS from radioiodide and P [125 I]T33 was h. Separation at P accomplished by Sephadex LH-20 chromatography. Average 25 1]I] yield from this method was 82 ± 3%. 3%. Authenticity Authenticity of of the the [1[125 (1 N N HCI HCl at at T 3S was verified in all instances by acid hydrolysis (1 80 C for 1 h) followed by LH-20 chromatography and HPLC analysis (2). Total serum T 44,, T 33,, and TSH were measured by previously (5). There was less described radioimmunoassay procedures (5). 2% cross-reactivity with T 33S in the T3 T 3 RIA and less than than 2% T4 RIA. 0.5% cross-reactivity with the T4 Statistical analysis was by paired t test with Bonnferoni Bonnferoni correction. Data presented as mean ± SEM.

Results Stability of f25JJT T^IJTsS 3S 251]T The stability of P [125 I]T3SS was determined by adding P [ 1]T I]T33SS to to serum serum and and urine and and storing storing aliquots aliquots for for 2 weeks at -20 C and 4 C, respectively. These aliquots were processed in the same manner as the patients' 25I] samples and demonstrated that less than 11% % of the [[1125 1] T 33S % in the urine underS in the serum and less than 22% went degradation. Further, the HPLC urinary profile 251]T [125 I]T3S showed that less than 11% % of the P 3S added to urine samples was converted to T33.. 125 25

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Protein binding of f25I]T / 125 //T3S After of After the addition of TCA to serum, 99.2 ± 5.1% 5.1% of the [1251]T [125I]T3SS present in the serum was precipitated in S circulates prethe protein fraction suggesting that T 33S finding dominantly bound to serum proteins (n = 8). This finding substantiated by equilibrium dialysis by comwas also substantiated paring the free fraction of tracer T 33S, S, T 44,, and T 3 after after S, T 44,, and 20 h of dialysis. The mean free fraction of T 33S, T3 T 3 were 0.009%, 0.009%, and 0.042%, respectively (n = 6). 125 Kinetics of serum serum /f25JJT Kinetics //T 3 S disposal

The MCR was estimated after after a single bolus injection injection as well as during the constant infusion infusion of [1251]T [125I]T33S. The after the bolus injection injection was 134.8 mean MCR calculated after = 7), which compared favorably with ± 15.2 L/day (n = the 127.2 ± 8.7 L/day (n = 3) value determined by constant infusion. The administration of PTU (300 mg [125I]T33SS compared to every 6 h) decreased the MCR of [1251]T control values obtained from the bolus studies (86.8 ± significantly decreased 8.9 L/D, P < 0.01). IA and fasting significantly 251]T MCR of P [125 I]T3S S compared to control (46.5 (46.5 ±± 9.8 9.8 L/ L/ the MeR compared to control 3 10.1 L/D, P < 0.002, respecD, P < 0.002 and 55.8 ± 10.1 tively). The various disappearance patterns are summarized in Fig. 1. There was an attempt to isolate tracer T 33 after tracer T 33S S was injected, but no tracer in the plasma after detected. T 33 could be detected. The acute temporal urinary excretion patterns of si251]T multaneously injected injected [1311]Na [131I]Na and P [125 I]T33SS are shown in Figs. 2 and 3. As shown in Fig. 2, the abrupt rise in 3. 125 131 the urinary P251]/[1311] ratio reflects the rapid intracel[ I]/[ I] 125 During the 3S. lular entry and deiodination of [1251]T [ I]T33 131 131 day collection period, the 90.6 ± 0.5% of the [ 1] I] and 25 1] [125 I] recovery in the urine resulted in a 83.4 ± 2.0% of P efficiency of 92.2%. 92.2%. The effects effects calculated deiodination efficiency of PTU (200 mg every hour for 6 h) administration on 251], [125 I], as shown in Fig. 3, the cumulative excretion of P reduced this this value value to to 45.8%. reduced

Relationship of deiodination inhibition to f25JJT 3S kinetics Figure 4 summarizes the estimated fractional urinary deiodination of [1251]T -D inhibition [125I]T33SS during during 5' 5'-D inhibition produced produced by PTU, lA, IA, and fasting. This was calculated as total radioiodine excreted in the urine divided by the dose of of 125 1251T3 IT3 injected injected (percent injected injected dose). dose). All three maneu25I] vers significantly 1] significantly reduced the total deiodination of [P125 T 33S, S. PTU administration (300 mg every 6 h) decreased /-Lg load deiodination to 69.7 ± 9.2%, P < 0.04, IA (500 fig and 500 ng /-Lg daily) diminished the deiodination to 25.9 ± 2.8%, P < 0.0001 and fasting (q (5 days) decreased the value 0.01, compared to control value of to 58.0 ± 6.2%, P < 0.01, of

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LOPRESTI ET AL. AL. LOPRESTI 100 IOO

JCE Mo •1991 1991 JCE & &M Vol73«No4 Vo173 o No4

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% STOlL STD/L 125IT3S disappearance FIG. 1. The serum 125 IT3S disappearance patterns for each study group are shown.

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FIG. 2. S estimated by 2. Deiodination efficiency efficiency of tracer T 33S by the the urinalY urinary 131 1]I]after 1]T and 1] I] excretion pattern of [1251] [125I] and and[1[31 afterinjection injectionofofP[25125 I]T and[131[181 3S 3S 31 25 Na is 1]/[1 1]I] is is shown. A A ratio of percent urinary [1[125 I]/[131 is plotted against time. time.

91.0 ± 1.7%. ±1.7%. The relationship of the reduced T 33S S deiodination fracfraction, shown as T 3S as percent of injected dose, dose, to to serum T3S clearance is is depicted in Fig. Fig. 5. 5. A A strong positive correlation between T 3S and urinary deiodination 3 S clearance and 0.65, P < 0.0006) was noted with IA IA adminfraction (r = 0.65, and fasting. In contrast, the the effect of PTU istration and on T T3S was less than that exadministration on 3 S clearance was for the the degree of inhibition of deiodination. deiodination. pected for 125 Urinary Urinary /f25J1T /VT3>S metabolite pattern pattern 3 S metabolite

The urinary metabolite pattern was was determined acutely after aa bolus injection as as well as as for for the the next 14

50 POST

1251 POST ORAL 1251 T3S 1251T3S 75

INJECTION

251] 3. The The appearance of P [12S I] in the the urine after the the iv iv injection of FIG. 3. of 25 I]T [125 I]T33SS with with (x) (x) and and without without (0) (O) PTU PTU administration (200 (200 mg mg every every P 25I]I] in h) in in depicted. depicted. Also, Also, the the appearance of P [125 in the the urine after the 6 h) 125 31 1] oral administration of [1251]T S (~) is shown. The [ I]T33 (A) is The appearance of [[1131 I] (O) after the injection of [131I]Na [131I]Na is is shown for for comparison. (0)

h after the the injection. A representative elution profile profile after a bolus injection of [125I]T [125I]T33S S isisshown shown in in Fig. Fig. 6.6.The The the [12 [1255I]T I]T3S major urinary peak comigrates with the 3S stand125 I]3,3'-T ard. Other peaks identified include [125 1]3,3' -T22S, small T 3 and and 3,3' 3,3 '-T as well well as as an an unidenunidenamounts of labeled T3 -T22 as tified peak. Agents inhibiting deiodination, such as as PTU PTU and lA, -T22S, 3,3' -T 22, IA, markedly reduced the the size of 3,3' 3,3 '-T 3,3'-T and the the unknown peaks (data not not shown). shown). Figure 7 depicts the methe elution profile of the the urinary thyronine metabolite pattern for for the the ensuing 14 14 h after the the bolus 125 injection of [125 1]T I]T33S. T3S T3S was was the the major metabolite identified, although its its percentage of the total thyronine thyronine fraction decreased with time. time. In contrast, both labeled and 3,3' 3,3'-T in which minimal amounts were initially initially -T 22,, in T 33 and time. detectable, gradually increased with time.

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CHARACTERISTICS OF T 3S METABOLISM IN IN EUTHYROID MAN

Discussion Discussion

•* P