Equilibrium dialysis studies of plasma binding of thyroxine, triiodothyronine and their glucuronide and sulfate conjugates in human and cat plasma

Binding of thyroxine (T4), triiodothyronine (T3) and their glucuronide and sulfate conjugates, T4G, T3G, T4S and T3S, was assessed by equilibrium dialysis in plasma from 6 euthyroid humans and 5 euthyroid cats. In humans, the dialyzable percentages of T4, T4G and T4S were 0.029 +/- 0.003 (mean +/- S.D.), 0.14 +/- 0.04 and 0.08 +/- 0.02 respectively. For T3, T3G and T3S they were 0.31 +/- 0.04, 0.33 +/- 0.07 and 0.22 +/- 0.02. In cats, the corresponding values for T4, T4G and T4S were 0.057 +/- 0.009, 0.20 +/- 0.10 and 0.53 +/- 0.04; for T3, T3G and T3S they were 0.47 +/- 0.08, 0.51 +/- 0.11, and 0.53 +/- 0.11. We conclude that glucuronide conjugation has little or no inhibiting effect on the plasma binding of T3, and that T3S is actually more tightly bound than T3 in human (but not feline) plasma. On the other hand, conjugation to either the glucuronide or the sulfate markedly reduced T4 binding in plasma from both species.     

Effects of propylthiouracil on the biliary clearance of thyroxine (T4) in rats: decreased excretion of 3,5,3'-triiodothyronine glucuronide and increased excretion of 3,3',5'-triiodothyronine glucuronide and T4 sulfate

The liver metabolizes T4 by deiodination and conjugation to T4 glucuronide (T4G), but little information exists about the formation of T4 sulfate (T4S) in vivo. We have examined the excretion of T4G, T4S, T3 and rT3 glucuronide (T3G and rT3G) in bile, collected under pentobarbital anesthesia 0-8 h or 17-18 h after iv [125I]T4 injection to control and 6-propyl-2-thiouracil (PTU)-treated rats. Radioactivity in bile, plasma, feces, and urine was analyzed by Sephadex LH-20 chromatography and HPLC. PTU induced a 2-fold increase in the biliary excretion of total radioactivity (26.6% vs. 15.0% dose between 0-8 h; 2.0% vs. 1.0% dose between 17-18 h). Biliary metabolites, 17-18 h after T4 injection, in control vs. PTU rats amounted to (percent dose): T4G, 0.44 vs. 0.75; T3G, 0.19 vs. 0.07; rT3G, 0.02 vs. 0.15; and T4S, 0.06 vs. 0.32. Similar results were obtained for control rats when bile was collected between 7-8 h after iv T4. The excretion rate of T3G was lower and that of rT3G higher when bile was continuously collected for 8 h immediately after T4 administration, probably due to prolonged experimental stress. However, regardless of the period of bile collection, PTU induced a more than 24-fold decrease in the T3G/rT3G ratio and a 5-fold increase in T4S excretion. In the animals killed 18 h after T4 injection, PTU treatment increased plasma T4 retention by 50%, reduced urinary I- excretion by 74%, and increased fecal radioactivity by 47%. No conjugates were detected in feces, and the distribution of fecal T4:T3:rT3 was 70:18:2 in control and 68:7:6 in PTU-treated rats. The results indicate that 1) the glucuronidative clearance of T4 is not affected by PTU; 2) the T3G/rT3G ratio in bile is a sensitive indicator of type I deiodinase inhibition; 3) T4 undergoes significant sulfation in rats in vivo, and 4) biliary excretion of T4S is enhanced if its type I deiodination is inhibited.     

Deiodination and deconjugation of the glucuronide conjugates of the thyroid hormones by rat liver and brain microsomes.

Radioiodinated thyroxine (T4) glucuronide (T4G) and triiodothyronine (T3) glucuronide (T3G), paired with T4 or T3, were incubated at 37 degrees C for 2 hours in the presence of dithiothreitol and microsomes that had been prepared from euthyroid rat liver or hypothyroid rat brain tissues, as sources of type I and type II iodothyronine 5'-deiodinases, respectively. Incubations with boiled microsomes served as controls. The incubated supernatant was analyzed by high-pressure liquid chromatography (HPLC) for content of T4, T4G, T3, T3G, and combined T2 and T2G. The deiodination of T4G resulted from incubation with both liver and brain microsomes, but was somewhat less active than the deiodination of simultaneously incubated T4. All batches of microsomes studied also caused deconjugation of both T4G and T3G. The data are compatible with the hypothesis that T4G can serve as an alternate pathway for conversion of T4 to T3 in these tissues.     

Enhanced thyroxine metabolism in hexachlorobenzene-intoxicated rats

The effect of hexachlorobenzene (HCB) (1 g/kg bw) administration for 4 weeks, on thyroxine (T4) and triiodothyronine (T3) metabolism was studied in Wistar rats. The effect on serum binding of T4 has also been studied. Animals were injected with a tracer dose of either labeled hormone and by examining serum L-125I-T4 and L-125-I-T3, kinetics of radiolabeled hormones metabolism were calculated. The T4 metabolic clearance (MCI) as well as the distribution space, were increased by 6 fold. Decreased serum T4 levels result from an increase both in deiodinative and fecal disposal in HCB-treated rats. 125I-T3 metabolism was slightly affected. The enhanced peripheral disposition of thyroxine appears to lead to increased thyroid function, as measured by augmented TSH serum levels and 125I-thyroidal uptake. Serum binding of T4 was not affected.     

Enterohepatic circulation of triiodothyronine (T3) in rats: importance of the microflora for the liberation and reabsorption of T3 from biliary T3 conjugates

In normal rats, T3 glucuronide (T3G) is the major biliary T3 metabolite, but excretion of T3 sulfate (T3S) is greatly increased after inhibition of type I deiodinase, e.g. with 6-propyl-2-thiouracil (PTU). In this study, the fate of the T3 conjugates excreted with bile was studied to assess the significance of a putative enterohepatic circulation of T3 in rats. Conventional (CV) or intestine-decontaminated (ID) rats received iv [125I]T3G or [125I]T3S, the latter usually after pretreatment with PTU (1 mg/100 g BW). Radioactivity in plasma and bile or feces was analyzed by Sephadex LH-20 chromatography and HPLC. Within 1 h, 88% of injected T3G was excreted in bile of CV or ID rats, independent of PTU. About 75% of the injected T3S was excreted within 4 h in PTU-treated rats, in contrast to only 20% in controls. Up to 13 h after iv administration of T3G or T3S (+PTU) to intact ID and CV rats, fecal radioactivity consisted of more than 90% T3 in all CV rats, 95% of T3S in T3S-injected ID rats, and 30% T3 and 67% T3G in T3G-injected ID rats. In overnight-fasted CV rats injected with T3G, total plasma radioactivity rapidly declined until a nadir of 0.10% dose/ml at about 2.5 h, but radioactivity reappeared with a broad maximum of 0.12% dose/ml between 5.5-10 h. In the latter phase, plasma radioactivity consisted of predominantly I- and T3 in a ratio of 2:1. Reabsorption was diminished in fed CV rats and prevented in ID rats. Plasma T3 4-10 h after iv T3G injection to overnight-fasted CV rats was 12, 2, and 3 times higher than that in bile-diverted rats, fed CV rats, and ID rats, respectively, and similar to that 4 h after the injection of T3 itself. Total plasma radioactivity as well as plasma T3 6-13 h after iv administration T3S in PTU-treated rats were significantly increased in CV vs. ID rats, e.g. T3 0.016% vs. 0.005% dose/ml. These results demonstrate a significant enterohepatic circulation of T3 in rats in which bacterial hydrolysis of T3 conjugates excreted with bile plays an important role.     

A case report of Hashimoto's disease with anti-thyroxine autoantibody (author's transl)]

In one case of untreated Hashimoto's disease, serum thyroxine (T4) value by radioimmunoassay (RIA) was significantly lower than that by competitive protein binding analysis (CPBA). The discrepancy was found to be due to the presence of antithyroxine autoantibody in the serum. This phenomenon was considered to be of practical importance in interpreting the T4 value by RIA in cases with autoimmune thyroid diseases. The patient was 59-year-old woman with a 30-year history of goiter. A diagnosis of Hashimoto's thyroiditis had been established by open biopsy of the thyroid ten years ago. The patient was judged to be euthyroid on the basis of clinical and laboratory evaluation (mean serum T4 by CPBA (Tetrasorb and Tetratab kit), 5.0 mug/100 ml; serum T3, 165 ng/100 ml; T3 resin uptake, 31.8%; and serum TSH, 2.0 muU/ml). TBG binding capacity was 24 mug/100 ml. Anti-thyroglobulin antibodies (anti-Tg), once positive ten years before, was negative at this time. But the mean T4 in the serum measured by T4 RIA and RIA-Mat T4 kit were 1.7 and 2.9 mug/100 ml, respectively. Recovery of the T4 added to the patient's serum evaluated by RIA-Mat T4 kit, was 71.2%, although the recovery using a control serum was 108%. Binding of 125I-T4 to the serum or fractions of the serum was studied by using polyethylene glycol (PEG) method, column chromatography, and double antibody precipitation. The results were as follows: 1) The binding of 125I-T4 to the patient's serum was detected by using RIA kit system without addition of anti-T4 serum. 2) On Sephadex G-200 chromatography of 125I-T4 incubated with the serum or the rabbit anti-T4 antibody in the presence of ANS, an early radioactive peak was observed by using the patient's serum as in the case of the anti-T4 antibody. When the serum after thermal inactivation of TBG, was incubated with 125I-T4, and was applied to the Sephadex G-200 column, a radioactive peak was observed in the area where 7S fraction was detected by protein peak. 3) The binding of 125I-T4 to the patient's IgG was 9.0% by using double antibody method when the binding to a control IgG was 0.5%. 4) The binding of 125I-T4 to IgG fractions was also proved by PEG method. 5) The binding of 125I-T4 was competitively inhibited by the addition of unlabeled T4. The affinity constant was 1.9 X 10(8) L/mol and its binding capacity was 0.8 mug/100 ml serum. From these data this T4 binding IgG was considered to be anti-T4 autoantibody. The cross reaction with T3 was approximately 8.3%. MIT and DIT did not displace labeled T4 when tested in amounts varying from 0.1 to 100 ng/assay. By using the paper electrophoresis, the binding of 125IT4 to the serum or IgG was not detectable. Therefore this method was considered unsuitable for detecting such anti-T4 antibody. As we couldn't find any significant binding of 125I-T4 to sera in 37 other patients with Hashimoto's disease by using the PEG method, the incidence of this phenomenon was considered to be low...     

Comparison of mechanisms mediating uptake and efflux of thyroid hormones in the human choriocarcinoma cell line, JAR

We compared the specificities of transport mechanisms for uptake and efflux of thyroid hormones in cells of the human choriocarcinoma cell line, JAR, to determine whether triiodothyronine (T3), thyroxine (T4) and reverse T3 (rT3) are carried by the same transport mechanism. Uptake of 125I-T3, 125I-T4 and 125I-rT3 was saturable and stereospecific, but not specific for T3, T4 and rT3, as unlabelled L-stereoisomers of the thyroid hormones inhibited uptake of each of the radiolabelled hormones. Efflux of 125I-T3 was also saturable and stereospecific and was inhibited by T4 and rT3. Efflux of 125I-T4 or 125I-rT3 was, in contrast, not significantly inhibited by any of the unlabelled thyroid hormones tested. A range of compounds known to interfere with receptor-mediated thyroid hormone uptake in cells inhibited uptake of 125I-T3 and 125I-rT3, but not 125I-T4. We conclude that in JAR cells uptake and efflux of 125I-T3 are mediated by saturable and stereospecific membrane transport processes. In contrast, the uptake, but not the efflux, of 125I-T4 and 125I-rT3 is saturable and stereospecific, indicating that uptake and efflux of T4 and rT3 in JAR cells occur by different mechanisms. These results suggest that in JAR cells thyroid hormones may be transported by at least two types of transporters: a low affinity iodothyronine transporter (Michaelis constant, Km, around 1 microM) which interacts with T3, T4 and rT3, but not amino acids, and an amino acid transporter which takes up T3, but not T4 or rT3. Efflux of T4 and rT3 appears to occur by passive diffusion in these cells.     

Simultaneous measurements of free thyroxine and triiodothyronine fractions in human serum by means of equilibrium dialysis (author's transl)]

Simultaneous measurements of serum free thyroxine (T4) and triiodothyronine (T3) fractions were studied using a modification of equilibrium dialysis described by Sterling and Brenner. To 1.2 ml of the serum to be assayed, 131I-T4 and 125I-T3 were added in a concentration of 2 mug/dl and 25 ng/dl, respectively, both of which were preliminarily dialysed according to Schussler and Plager. Half ml of the serum with tracers added was dialysed against 9 ml of phosphate buffer (ionic strength 0.15, pH 7.4) for 18 hours at 37 degrees C and 0.1 ml was reserved from the rest in a counting tube (in duplicate). After the completion of dialysis, the dialysate was mixed with 1 ml of pool serum and the contaminating inorganic iodide (in the form of 131I or 125I) was eliminated by adsorption on anion exchange resin. The radioactivity of 3 ml of the dialysate and 0.1 ml of the preserved serum (with the tracers added) was counted and the free (or dialysable) fractions were expressed as a ratio of the count of the former divided by that of the latter adjusted to an equal volume by calculation. The amount of either T4 or T3 added as tracers had no influence on free T4 or T3 fraction unless either of them was added to a concentration of 10 mug/dl. When 125I-T3 of low specific activity (50 muCi/ug) was used as a tracer, free T3 fraction measured simultaneously with free T4 fraction tended to be higher than that measured with a single tracer. When 125I-T3 of higher specific activity (300 muCi/mug) was employed, free T3 fraction obtained with two methods did not differ significantly. Using serum T4 and T3 concentrations measured by competitive protein binding analysis and radioimmunoassay, respectively, the free T4 and T3 concentrations were estimated with sera of normal, hyperthyroid, hypothyroid and uncomplicated pregnant subjects.     

INHIBITION OF CONVERSION OF THYROXINE TO TRIIODOTHYRONINE IN PATIENTS WITH SEVERE CHRONIC ILLNESS

Many clinically euthyroid patients with severe, chronic, non-thyroidal illnesses (i.e. sick euthyroid patients) have very low circulating concentrations of total and absolute free triiodothyronine (T3), low-normal concentrations of total thyroxine (T4), elevated concentrations of absolute free T4, and circulating concentrations of thyrotrophin (TSH) that are either normal or subnormal. This study was undertaken to elucidate the mechanism of the low circulating T3 concentrations. The disappearance rate of ¹²⁵I-T3 from the circulation of five representative sick euthyroid patients, was studied and found to be slower, but not significantly so, compared with three control subjects, thus excluding an increased destruction rate as the cause of the low T3 levels. A selective decrease of T3 secretion from the thyroid gland of these patients was also excluded by the results of TSH stimulation tests. Inhibition of extra-thyroidal conversion of T4 to T3 was suggested by studies of the thyroid function in a hypothyroid woman with a Grade IV lymphoma on T4 replacement therapy. When the lymphoma was in remission, her circulating T3 concentration was 2–55 nmol/1 but when it relapsed it fell to 0–55 nmol/1. The T4 concentrations were 124–7 nmol/1 and 126 nmol/1 respectively. Decreased monodeiodination of T4 to T3 in sick euthyroid patients was confirmed by paper chromatography of extracted serum obtained 48 h after an i.v. injection of ¹²⁵I-T4 into two severely ill patients from the intensive therapy unit and a control subject. Peaks of radioactivity corresponding to ¹²⁵I-T4 and ¹²⁵I-T3 were detected in the control subject, but only a single peak corresponding to ¹²⁵I-T4 was detected in the ill patients.     

Anti-triiodothyronine antibodies in patients with rheumatoid arthritis associated with Hashimoto's thyroiditis

Anti-triiodothyronine antibody was found in a case of rheumatoid arthritis associated with Hashimoto's thyroiditis. The patient was a 40 year-old woman who had complained of polyarthralgia, joint-swelling and stiffness for seven years. She had a rheumatoid nodule and showed a positive RA test. Radiographic changes of hands and wrists showed osteoporosis, erosions and narrowing of joint space. Nonsteroidal anti-inflammatory drugs had been used for seven years. The diagnosis of Hashimoto's thyroiditis had been made by open biopsy of the thyroid gland seven years before. Serum T4, TSH, TBG, free T4, free T3 and r-T3 were all normal. On the other hand, serum T3 level was almost unmeasurable by radioimmunoassay. Binding of 125I-T3 to the patient's serum was studied by using polyethylene glycol (PEG) and column chromatography. By using the PEG method, the binding of 125I-T3 to the patient's serum was tenfold compared to control serum. Sephadex G-25 column chromatography (0.9 X 1.5 cm) of 125I-T3 with the patient's serum in the presence of 0.1% ANS showed an early radioactive peak, while control serum did not show an early peak. In the next experiments, the patient's serum was labelled with 125I-T3, mixed with human anti-IgG, IgM, IgA, lambda, kappa, incubated at 4 degrees C for 20 hours and centrifuged for 20 min. Strong binding to the anti-IgG and anti-lambda was detected. The presence of this abnormal T3-binding globulin in the patient's serum may have produced an undetectable T3 level.     

Thyroxine and 3,3',5-triiodothyronine are glucuronidated in rat liver by different uridine diphosphate-glucuronyltransferases

Male Wistar rats were treated with 50 mg 3,3',4,4'-tetrachlorobiphenyl (TCB)/kg BW or vehicle. After 4 days, the livers were isolated and perfused for 90 min with 2 nM [125I]T3 or 10 nM [125I]T4 in Krebs-Ringer medium containing 1% albumin. Deiodination and conjugation products and remaining substrates were determined in bile and medium samples by Sephadex LH-20 chromatography and HPLC. TCB treatment did not affect hepatic uptake and metabolism of T3. However, biliary excretion of T4 glucuronide was strongly increased by TCB, resulting in an augmented T4 disappearance from the medium, although initial hepatic uptake of T4 was not altered. Measurement of the microsomal UDP-glucuronyltransferase (UDPGT) activities confirmed that T4 UDPGT was induced by TCB, whereas T3 glucuronidation was unaffected. T3 UDPGT activity showed a discontinuous variation, which completely matched the genetic heterogeneity in androsterone glucuronidation in Wistar rats. These results indicate that different isozymes catalyze the glucuronidation of T3 and T4.     

Metabolism of triiodothyroacetic acid (TA3) in rat liver. II. Deiodination and conjugation of TA3 by rat hepatocytes and in rats in vivo

The hepatic metabolism of 3,3',5-triiodothyroacetic acid (TA3), a naturally occurring side-chain analog of T3, was studied in vitro and in vivo. Metabolites were quantified by HPLC after Sephadex LH-20 prepurification of samples obtained after incubation of [125I]TA3 or 3,[3'-125I]diiodothyroacetic acid (3,[3'-125I]TA2) with isolated rat hepatocytes under various conditions or after iv administration of [125I]TA3 to normal or 6-propyl-2-thiouracil (PTU)-treated rats. In protein-free incubations with hepatocytes, TA3 glucuronide (TA3G) and I- were normally the main TA3 products, i.e. 44% and 49%, respectively. In the presence of the type I deiodinase inhibitor PTU, the I- production from added TA3 decreased to 3%, and TA3 sulfate (TA3S) increased from 2-14%. Normally, 3,3'-TA2 was converted to I-, but in the presence of PTU 3,3'-TA2S was produced. In SO4(2-)-depleted cultures incubated with TA3 or 3,3'-TA2, production of I- was diminished, and the glucoronides of the substrates and the deiodinated products were generated. If both sulfation and deiodination were inhibited, TA3 and 3,3'-TA2 were cleared completely via glucuronidation. The metabolism of TA3 and especially 3,3'-TA2 was greatly retarded in cultures with 0.1% BSA. PTU treatment of TA3-injected rats reduced plasma I- levels 6-fold, increased plasma sulfates 2.6-fold, but did not affect plasma TA3 clearance. Biliary excretion of radioactivity until 4 h after [125I]TA3 injection amounted to 55% of the dose in controls vs. 85% in PTU-treated rats. In both groups, an unknown metabolite X was detected in serum and its sulfate conjugate XS in bile. The mean percent distribution of TA3G/TA3S/XS in bile amounted to 70:8:13 in control and 57:22:12 in PTU rats. In conclusion, TA3 is effectively metabolized in rat liver by glucuronidation and subsequent biliary excretion of TA3G, which may explain its rapid in vivo clearance relative to T3. Furthermore, a significant proportion of TA3 is deiodinated by the type I deiodinase, either directly or after prior sulfation.     

Identification of 3,3'-diiodothyroacetic acid sulfate: a major metabolite of 3,3',5-triiodothyronine in propylthiouracil-treated rats

The sulfate conjugate 3, [3'-125I] diiodothyroacetic acid (3,3'-TA2S) was discovered in plasma, and occasionally in bile, of 6-propyl-2-thiouracil-treated rats after administration of [125I]T3. The identification of this T3 metabolite was based on the following evidence: 1) the compound co-eluted in two different HPLC systems with synthetic 3,3'-TA2S; 2) its chromatographic behavior on Sephadex LH-20 was characteristic for a conjugated iodothyronine derivative; and 3) the metabolite was hydrolyzed by arylsulfatase and the liberated product comigrated with synthetic 3,3'-TA2 on HPLC. Marked accumulation of 3,3'-TA2S was observed only in rats with impaired type I deidodinase activity but not in controls. Furthermore, plasma and biliary 3,3'-TA2S levels varied with the experimental conditions such as anesthesia, i.e. both were increased in ketamine-anesthetized over pentobarbital-anesthetized animals. It was not possible to indicate the exact pathway through which 3,3'-TA2S is generated from T3; neither is it known how much of T3 is actually metabolized via 3,3'-TA2S. However, the significant plasma 3,3'-TA2S levels, even in unanesthetized animals, illustrate the physiological relevance of this T3 metabolite.     

Triiodothyronine(T3) autoantibodies in a woman with nonthyroidal disorder: a study on preparation of serum IgG fraction employing protein A column chromatography

A 48-year-old non-goitrous woman, who had undergone cardiac surgery for mitral stenosis under the extracorporeal circulation, showed high levels of serum T3 and free T3 in a recent follow-up study, employing antibody coated-bead RIA for T3 and -Amerlex M particle RIA for free T3. However, other thyroid function tests (T4, free T4, TSH and TBG) were normal. We suspected that thyroid hormone autoantibodies (THAA) in her serum interfered with T3 and free T3 analyses. The presence of THAA was demonstrated by the use of various procedures as follows. Firstly, the patient's serum was directly incubated with 125I-T3 or -T4 analog which did not bind to TBG, followed by B/F separation with polyethyleneglycol, counting the precipitates. Secondly, after the serum was treated with an acid-charcoal solution to remove circulating thyroid hormone, the measurement of THAA was made as stated above. Normal sera were used as controls. Both the non- and acid-charcoal-treated sera showed much higher percentages of 125I-T3 analog precipitation as compared with controls. In the case of 125I-T4 analog, there was no difference between them. In the third study, the presence of IgG antibodies that bound T3 but not T4 was investigated. The IgG fraction of the patient's serum was separated employing a Protein A-Sepharose CL-4B column chromatography. Then, the prepared IgG fraction was purified by a technique of gel filtration chromatography (Sephacryl S 200). Non-purified and purified-IgG fractions both revealed higher binding percentages of 125I-T3 analog than the control IgG fraction and non-IgG fraction of the patient. Furthermore, a good dose response was observed between the binding percentage of 125I-T3 analog and each dose of the patient's serum or IgG fraction. From these observations, it was clarified that this woman had anti-T3 IgG autoantibodies using a Protein A column chromatography with confirmation of gel filtration chromatography.     

Metabolism of triiodothyronine in rat hepatocytes

The metabolism of T3 by isolated rat hepatocytes was analyzed by Sephadex LH-20 chromatography, HPLC, and RIA for T3 sulfate (T3S) and 3,3'-diiodothyronine (3,3'-T2). Type I iodothyronine deiodinase activity was inhibited with propylthiouracil (PTU), and phenol sulfotransferase activity by SO4(2-) depletion or with competitive substrates or inhibitors. Under normal conditions, labeled T3 glucuronide and I- were the main products of [3'-125I]T3 metabolism. Iodide production was decreased by inhibition (PTU) or saturation (greater than 100 nM T3) of type I deiodinase, which was accompanied by the accumulation of T3S and 3,3'-T2S. Inhibition of phenol sulfotransferase resulted in decreased iodide production, which was associated with an accumulation of 3,3'-T2 and 3,3'-T2 glucuronide, independent of PTU. Formation of 3,3'-T2 and its conjugates was only observed at T3 substrate concentrations below 10 nM. Thus, T3 is metabolized in rat liver cells by three quantitatively important pathways: glucuronidation, sulfation, and direct inner ring deiodination. Whereas T3 glucuronide is not further metabolized in the cultures, T3S is rapidly deiodinated by the type I enzyme. As confirmed by incubations with isolated rat liver microsomes, direct inner ring deiodination of T3 is largely mediated by a low Km, PTU-insensitive, type III-like iodothyronine deiodinase, and production of 3,3'-T2 is only observed if its rapid sulfation is prevented.     

Studies on thyroid hormone autoantibody (THAA) in 2 cases of Graves' disease with spuriously high free thyroxine values

Two patients with Graves' disease treated with methimazole (MMI) showed a discrepancy between serum free T4 (FT4) values and other hormone values (especially total T4) which was due to the presence of potent binding activity to labelled T4 analogue (125I-aT4) in their serum. This activity was demonstrated to be in immunoglobulin G (IgG) with kappa light chain isotype in both patients. The binding of 125I-aT4 to their serum was inhibited by unlabelled T4 in a dose-dependent manner. Autoantibodies had almost identical binding affinity to T4 and aT4, although they precipitated more radioactivity when 125I-aT4 was used. The binding of IgG purified from patients' sera to labelled T4 or aT4 was not greater than the corresponding sera, suggesting that the thyroxine binding proteins did not interfere with the assay. Since the specific radioactivity of 125I-aT4 is almost 10 times higher than that of 125I-T4, autoantibodies can precipitate almost 10 times more radioactivity in the FT4 assay than the total T4 assay, thus leading to the spuriously high FT4 values and large discrepancy between FT4 and TT4 values.     

Thyroid hormone binding to adult rat alveolar type II cells. An autoradiographic study

Hypothyroid adult rats were injected intravenously with [3, 5, 3'-125I]triiodothyronine (T3) or [125I]thyroxine (T4). Others were similarly treated except for the addition of a 500-fold excess of the appropriate unlabeled hormone. Light microscopic autoradiography of semithin sections of lung from these animals demonstrated labeling localized over alveolar parenchymal tissue. Analysis of the labeling studies of type II cells revealed high affinity, low capacity binding of T3 and, to a much lesser extent, T4 to both the nuclear and cytoplasmic compartments. Adult lung in organ culture was also treated with 125I-T3 or 125I-T4 and electron microscopic autoradiography performed. These studies revealed that, when expressed as grains per square micrometer, there was substantially more labeling over the lamellar inclusion bodies and the mitochondria than over the nucleus. The results of this study provide morphologic evidence of specific uptake and binding of thyroid hormones by the nucleus and cytoplasm of alveolar type II cells, and suggest that the lamellar inclusion bodies and mitochondria may be the primary location of the cytoplasmic binding. These findings also substantiate the view of a direct effect of thyroid hormones on the lung and, in particular, on the type II alveolar cells.     

AUTOANTIBODIES TO THYROGLOBULIN CROSS REACTING WITH IODOTHYRONINES

Serum thyroxine was consistently immeasurable by radioimmunoassay in an elderly patient with myxoedema after successful treatment with oral thyroxine. Abnormal binding of thyroxine was suspected and shown to be due to the presence in serum of antibodies of the IgG variety. The characteristics of these antibodies with respect to their binding of thyroxine (T4), triiodothyronine (T3), reverse triiodothyronine (rT3) and human thyroglobulin (Tg) were systematically studied. Three preparations of Tg, and T4, T3 and rT3 were examined for their ability to compete with ¹²⁵I-Tg, ¹²⁵I-T4, ¹²⁵I-T3 and ¹²⁵I-rT3 for binding to the antibodies. For each tracer used the order of competitive efficiency was Tg > T4 > T3 > rT3. This provides for the first time direct evidence that iodothyronine reacting antibodies occurring in man are generated against Tg. All three iodothyronines were able to inhibit tracer binding of labelled iodothyronines completely, the order of effectiveness being T4 > T3 > rT3, suggesting antibodies with one type of binding site and that these were probably raised against a Tg sequence incorporating T4, although there was some evidence for the existence of a minor subpopulation of antibodies with higher specificity for T3. Complete displacement of labelled Tg by cold iodothyronines, however, was not possible. The experimental evidence suggests two classes of Tg antibodies, 70% of which were directed towards the T4 containing region, and 30% directed against other part(s) of the Tg molecule. Despite the presence of such Tg antibodies conventional haemagglutination tests of the patient's serum for Tg antibodies were negative.     

Intrathyroid deiodination of thyroxine: the effect of thyrotropic hormone and denervation of the thyroid

Intrathyroid transformation of thyroxin (T4) into triiodothyronine (T3) and the role of thyrotropic hormone and denervation in this process were studied with in vivo blood perfusion of dog thyroid gland in situ and at 37 degrees C. Both labelled 125I-T4 (200,000 imp/min) and nonlabeled T4 (1000 ng) were administrated in the arteries of thyroid lobules in situ and in a constant-temperature cabinet. Enhanced excretion of triiodothyronine (both labeled and nonlabeled) observed two hours after the injection in the thyroid blood output was suggested as an evidence of denervation-independent deiodination occurring in the thyroid tissue. Without T4 load the proportion of T3 in the thyroid venous blood was significantly increasing under the thyroid denervation thus attesting inhibitory effect of nervous impulse on T4 deiodination in the thyroid gland. TTH being administrated into thyroid arteries in a dose of 20 I.U. produced a pronounced elevation of T4 thyroid conversion in T3 which was less manifest in the process of denervation thus evidencing the high sensitivity of the process to TTH.     

Change in serum and biliary esterified bile acids in patients with extrahepatic cholestasis during percutaneous transhepatic cholangiodrainage

Eighteen patients with total extrahepatic cholestasis undergoing PTCD were classified into three groups, depending on the bilirubin decrease rate at two weeks after PTCD. Serum and biliary esterified bile acids in each group were measured before PTCD and at 24 hours, 48 hours, 1 week, and 2 weeks after PTCD. Bile acids were measured by Okuyama's methods (HPLC), and esterified bile acids were calculated from the difference between samples treated with sulfatase or beta-glucuronidase for enzymatic hydrolysis and untreated samples measured at the same time. The following results were obtained. The percentages of biliary esterified bile acids in total bile acids were as follows: before PTCD, in the fair improvement group, sulfate (S) = 6.4 +/- 4.6% (mean +/- S.D.), glucuronide (G) = 11.7 +/- 9.0%; in the poor improvement group, S = 2.8 +/- 1.6%, G = 1.0 +/- 0.9% and at 24 hours after PTCD, in the fair group, S = 9.1 +/- 7.5%, G = 7.5 +/- 4.3%; in the poor group, S = 2.9 +/- 2.4%, G = 1.7 +/- 1.1%. The percentages of esterified bile acids in the fair group were higher than in the poor group, and significant differences were noted in G (p less than 0.05). Thus PTCD is expected to reduce jaundice in cases with high percentages of biliary esterified bile acids before and shortly after PTCD.