Degradation Of rT3 Research Articles

Hormone-specific alterations of T4, T3, and reverse T3 metabolism with recent ethanol abstinence in humans.

Effects of recent alcoholic withdrawal on thyroxine (T4), 3,5,3'-triiodothyronine (T3), and reverse T3 (rT3) metabolism were determined by serum tracer kinetic studies in recently abstinent alcoholics without overt hepatocellular injury or caloric deprivation. Data were compared with those of normal subjects using a three-pool model, with rapidly and slowly equilibrating pools exchanging with serum. Significant differences included 1) reduced serum total rT3 levels (to 69% of normal) and rT3 degradation rates (to 61%); 2) increased rT3 binding in rapidly (to 557%) but reduced binding in slowly (to 13%) equilibrating tissues, with opposite effects on rT3 fractional transfer rates to serum from rapidly (to 7.5%) and slowly equilibrating sites (to 669%); 3) increased T4 fractional transfer rates from serum to rapidly equilibrating tissues (to 122%); and 4) increased T4 binding to both rapidly (to 195%) and slowly (to 190%) equilibrating tissues. T3 kinetics were not significantly altered. Thus recently abstinent alcoholics have hormone-specific alterations of T4, T3, and rT3 transfer, distribution, and metabolism distinct from other nonthyroidal illnesses or caloric deprivation. Furthermore, these data indicate separate transfer processes for T4, T3, and rT3 from serum to tissue sites and hormone-specific tissue binding characteristics in humans in vivo.

Disposal and distribution of rT3 in humans: a new double-tracer kinetic study

A satisfactory definition of reverse 3,3',5'-triiodothyronine (rT3) kinetics in humans cannot be obtained if the plasma disappearance curve of the injected labeled hormone is the only experimental data available; most of the kinetic parameters can only be bounded within ranges showing unacceptable variabilities. To gain additional experimental data a double-tracer approach is proposed. After simultaneous injection of [125I]rT3 and 131I the following three experimental curves were determined in plasma: 1) the disappearance of [125I]rT3, 2) the disappearance of 131I, and 3) the appearance of 125I generated in vivo from labeled rT3 degradation. Combined analysis of these three curves, based on a complex six-compartment model, was developed and applied to data obtained in a group of normal subjects. Through this new analysis, fractional disposal rates and fractional exchange rates between the plasma compartment and the periphery are uniquely determined. The main physiologically interesting information on the degradation of the hormone that emerges from these studies are 1) all degradative pathways of rT3 generate iodide, being in all cases the [125I]rT3 dose completely recovered as 125I in plasma; and 2) most rT3 is degraded (65-90%) in peripheral tissues rapidly exchanging with the plasma pool. The extended experimental base is not yet sufficient to compute unique values for production rate (PR) and body mass (Qt); the validity of estimates of PR and Qt is based on the assumption that injected [125I]rT3 is able to trace all rT3 peripherally produced (from thyroxine). The new approach yields ranges for PR and Qt (1.12-2.15 micrograms/h and 2.88-8.24 micrograms) much narrower than those computable from the [125I]rT3 disappearance curve only (1.12-5.07 micrograms/h for PR and 2.88-23.7 micrograms for Qt).

RT3 metabolism in patients with nephrotic syndrome and normal GFR compared with normal subjects

To evaluate reverse 3,3',5'-triiodothyronine (rT3) metabolism in nephrotic syndrome, serum rT3 kinetic studies from 10 nephrotics (mean urinary protein losses 7.0 g/day) with normal glomerular filtration rates (GFR; creatinine clearance 107 ml/min) were compared with 9 normal healthy subjects. Serum disappearance data were analyzed in a three-pool model, including rapidly (liver and kidney) and slowly (muscle, skin, and brain) equilibrating pools exchanging with serum, with all losses from the rapidly equilibrating pool. Serum free thyroxine (T4), determined by equilibrium dialysis, and parathyroid hormone levels were unaltered; total T4, T3, and rT3, and free rT3, albumin, and transferrin levels were significantly decreased; and free fractions of T4 and rT3 and thyroid-stimulating hormone (TSH) levels were increased. Despite reduced rT3 binding in serum, fractional transfer rates from serum to extravascular sites and serum clearance rates of total rT3 were unaltered. Free hormone clearance, serum appearance, and maximum hormone production rates were decreased. Total hormone transfer rates between serum and tissue pools and rT3 mass in serum and both tissue pools were reduced. Binding in the slowly equilibrating pool was decreased, and binding in both rapidly and slowly equilibrating pools was correlated with the free fraction of rT3 (r = -0.79, P = 0.007, and r = -0.70, P less than 0.025, respectively), with a shift of rT3 from the slow to the rapid pool. These findings suggest that binding of rT3 and T4 to serum carrier proteins is reduced, the transfer process for rT3 from serum to extravascular sites is decreased by factors in addition to reduced serum binding, degradation of rT3 is impaired, and decreased slow-pool binding may reflect reduced rT3 binding to serum-derived proteins in interstitial fluid. Furthermore, rT3 production rates are reduced, despite normal serum free T4 levels, accounting for low serum free rT3 concentrations. Total rT3 levels are decreased because of decrements in both serum binding and production rates.

Thyroxine inner ring monodeiodinating activity in fetal tissues of the rat.

We studied thyroxine (T4) inner ring monodeiodinating activity (5-MA) in various tissues of fetal, maternal, and adult male rats. Tissue homogenates were incubated with 0.26 microM T4 in 0.1 M phosphate buffer (pH 7.4) containing 10 mM EDTA and 400 mM dithiothreitol (final volume 0.7 ml) for 10 min at 37 degrees C; the 3,3',5'-triiodothyronine (rT3) generated was measured by radioimmunoassay of ethanol extracts of incubation mixture and the result was corrected for rT3 degradation during incubation. Compared to maternal tissues, T4 to rT3 5-MA in the 14-day-old fetus was increased about 70 times in skeletal muscle (mean +/- SEM, velocity, 5.4 +/- 0.9 versus 0.08 +/- 0.01, pmol rT3/h/mg protein); approximately 8 times in intestine (0.72 +/- 0.17 versus 0.09 +/- 0.03);and approximately 4 times in cerebral cortex (19 +/- 0.5 versus 4.5 +/- 0.9), while it was similar in skin (3.2 +/- 0.48 versus 2.6 +/- 0.52). Hepatic T4 5-MA approximated 1.1 +/- 0.63 in the 14-day-old fetus; it could not be measured reliably in maternal or 19-day fetal tissue because of extensive (greater than 90%) degradation of rT3 during incubation. Relative to mother, T4 5-MA in 19-day fetal tissues was increased approximately 30-fold intestine, approximately 20-fold in skeletal muscle, and approximately 6-fold in cerebral cortex while it was similar in skin. The T4 5-MA in maternal rat tissues did not differ significantly from corresponding values in adult male rat, except skin, where it was lower in the mother rat (2.6 +/- 0.52 versus 4.6 +/- 0.61, p less than 0.05). In summary, relative to adult tissues T4 5-MA is exceedingly active in several fetal tissues, most notably in skeletal muscle followed by intestine and cerebral cortex.

Nuclear reverse T3 binding sites: an artefact of isolation?

Binding of [125I]rT3 to rat liver nuclear extracts prepared in 0.25 M sucrose could be abolished by a prior wash of the nuclei with 2.4 M sucrose. Analysis of mixtures containing [125I]rT3 and nuclear extracts (0.25 M sucrose) showed that after incubation for 2 h at 22 degrees C, degradation of rT3 into 3,3'-T2 and I- had taken place. It appears that the presence of 125I- in these mixtures can account for the previously observed 'binding' of [125I]rT3 to these nuclear extracts. Further characterization of the deiodination process in nuclear extracts showed: 1) inactivation by heating, 2) production of equimolar amounts of I- and 3,3'-T2, 3) stimulation by sulfhydryl compounds and inhibition by propylthiouracil in a fashion similar to the microsomal iodothyronine 5'-deiodinase (ping-pong mechanism). We conclude that the observed deiodination of rT3 in hepatic nuclear extracts is of enzymatic nature, due to contamination of the nuclear preparation by microsomal iodothyronine 5'-deiodinase. However, since the nuclei are prepared in the presence of the non-ionic detergent Triton X-100, a nucleus associated deiodinase activity cannot be totally excluded.

Preincubation of thyroxine with sulfhydryl-reducing agents does not stimulate thyroxine inner or outer ring deiodination.

Sulfhydryl reagents stimulate enzymatic conversion of T4 to T3 and rT3. A recent study suggested that such reagents stimulated T4 5'-deiodination by a direct interaction with T4. We therefore tested the ability of dithiothreitol (DTT) and other sulfhydryl reagents to enhance the susceptibility of T4 and rT3 to 5'-deiodination by liver homogenates and of T4 to 5-deiodination by placental homogenates. Preincubation of T4 with DTT in concentrations ranging from 0.5-80 mM did not result in increased T3 production from T4 in rat liver homogenates, nor was T3 production increased by preincubation of T4 with reduced glutathione or mercaptoethanol. Preincubation of rT3 with DTT also did not result in increased rT3 degradation by liver homogenates. T4 5-deiodination to rT3 by rat and human placental homogenates was not consistently increased by preincubation of T4 with DTT in concentrations ranging from 2.25-450 mM. These results do not support the hypothesis that sulfhydryl stimulation of T4 deiodination occurs as a result of sulfhydryl-T4 interaction.

Monodeiodination of thyroxine to 3,3', 5-triiodothyronine and 3,3', 5'-triiodothyronine in human kidney homogenate (author's transl)

The renal cortex, later proved by histology to be free of carcinoma, was obtained from the kidneys of 9 patients undergoing nephrectomy for hypernephroma or ureteral carcinoma. The cortex was homogenized in a cold 50mM Tris-HCl buffer, pH 7.5, and centrifuged at 800 X g. Supernatants (1.8 mg protein, referred to as "homogenate") were enriched with 2 micrograms of T4 and were incubated for varying periods at different temperatures. The T3 and rT3 formed were extracted into ethanol and measured by RIA. Reaction mixtures contained 5mM dithiothreitol, without which formation of T3 and rT3 from T4 was negligible. The production of T3 and rT3 from T4 was abolished by prior heating of the homogenate to 56 degrees C for 30 min. In fresh homogenates, the production of T3 and rT3 increased with an increased concentration of homogenate (0.2-1.8 mg protein), increased incubation temperature up to 37 degrees C, increased incubation time up to 60 min, and increased T4 concentration up to 8 micrograms/tub. T3 production from T4 was near maximal at pH 6.5 and rT3 production at pH 10. At the standard pH of 7.5, rates of net T3 and rT3 production were 58 and 45%, respectively, of those at the optimum pH. Degradation of rT3 was rapid, while degradation of T3 was negligible. Both T3 and rT3 production from T4 were inhibited in a dose dependent manner by ipodate, propylthiouracil and salicylate. The apparent Km values for monodeiodination of T4 to T3 was 10 microM. Among the usual subcellular fractions of the kidney homogenate, microsomes were most potent in deiodinating T4 to T3 and to rT3. These results indicate that the human renal cortex contains the enzymes generating T3 and rT3 from T4.

Monodeiodination of thyroxine to 3,3',5'-triiodothyronine in the human placenta

We investigated the characteristics of the monodeiodination of thyroxine to T3 and rT3 in human placentas which were obtained at normal delivery. The placentas were homogenized in a cold sucrose Tris-HCl buffer, pH 7.5. The microsomal fraction was incubated at 37 degrees C in air for 1 hr with 2 micrograms of T4 in the presence of 50mM DTT. The T3 and rT3 generated in the reaction mixture were extracted into cold ethanol and measured by RIA. Among the usal subcellular fractions of the placental homogenate, microsomes were the most potent in deiodinating T4 to rT3. In microsomes, production of rT3 increased with protein concentration, incubation temperature up to 37 degrees C, incubation time up to 120 min and T4 concentration up to 16 micrograms/tube. The production of rT3 from T4 was lost by prior heating of the microsomal fraction to 56 degrees C for 30 min. The net production rate of T4 to rT3 in the microsomal fraction was 17.9 ng/mg protein/micrograms T4/60 min at pH 7.5. RT3 production from T4 was maximal at pH 7.0. The production of T3 from T4 was negligible in the present system. Degradation of T3 in the placentas was rapid. Although the addition of anti-T3 antibody to the reaction mixture suppressed the degradation of T3, it had no effect on the net production of T3, suggesting that the obtained net T3 production rate had not been influenced by its degradation. Degradation of rT3 was negligible. These results indicate that the human placenta actively deiodinates T4 to rT3 enzymatically. This enzyme system might have some influence on the transplacental passage of the thyroid hormone from the mother to the fetus.

Effects of an inhibitor of hepatic drug metabolism, 2-diethylaminoethyl-2,2-diphenylvalerate HCl (SKF 525-A), on thyroxine metabolism in the rat.

We have examined some effects of 2ndash;diethylaminoethyl- 2,2-diphenylvalerate HC1 (SKF 525-A), an inhibitor of hepatic drug metabolism and of cytochrome P-450-mediated reactions, on T4 metabolism by rat liver slices and by rats in vivo. Incubation of liver slices with labeled T4 in medium containing 0.1 mM SKF 525-A reduced deiodination to 27% of control and did not affect T3 formation; at 1.0 mM SKF 525-A, deiodination and T3 formation both declined (to 7% and 60% of control levels, respectively), while degradation of rT3 was not affected. Hypoxia had effects similar to SKF 525-A, i.e. inhibition of T4 deiodination, while formation of T3 from T4 and degradation of rT3 were not inhibited. During a constant infusion of [125I]T4, SKF 525-A (3.5 mg/100 g BW, ip) caused an acute increase in the MCR of T4. It was then found that SKF 525-A added to serum in vitro increased the free T4 fraction; injected in vivo, it caused a prompt rise in the free T4 fraction accompanied by a fall in serum TSH. In rats receiv...

A study of the properties of the enzyme in rat liver that deiodinates 3,3'5'-triiodothyronine to 3,3'-diiodothyronine.

Studies were performed to assess the factors that influence the metabolism of [125I]rT3 in homogenates of rat liver. [125I]rT3 was metabolized with great rapidity; in 10% homogenates, rT3 was completely degraded within 30 min, giving rise to nearly equal proportions of two 125I-labeled metabolites, [125I]3,3′-diiodothyronine ([125I]3,3′-T2) and [125I]iodide. This process appeared to be enzyme mediated, since it was heat labile, temperature dependent, concentration dependent, and saturable with stable rT3. Aerobic preincubation markedly impaired the reaction, while anaerobic preincubation did not. These effects appeared to be due, respectively, to depletion and preservation of tissue glutathione (GSH) content during preincubation. Thus, the loss of activity seen after aerobic preincubation was prevented by the addition of either GSH itself or a NADPHgenerating system. GSH (5 HIM) stimulated [125I]rT3 degradation in dilute homogenates (1–5%) but not in concentrated homogenates (10–20%). Further evidence sug...

Serum Concentrations, Metabolic Clearance Rates, and Production Rates of Reverse Triiodothyronine, Triiodothyronine, and Thyroxine in Starved Rabbits*

It has been reported that in fasted human subjects, serum 3,3′,5′-triiodo-L-thyTonine (rT3) concentrations increase and serum 3,5,3′-triiodo-L-thyronine (T3) levels decrease without any significant change in serum L-thyronine (T4) and TSH concentrations. However, serum hormone concentrations may be affected by both the production and the degradation of the hormone. Studies were performed to determine serum concentrations, metabolic clearance rates (MCR) and production rates (PR) of rT3, T3, and T4 in starved rabbits. Serum T3 and T4 concentrations were determined by commercial kits, and serum rT3 levels were measured by the specific RIA developed in our laboratory. The MCR of T4 was determined by single compartmental and those of T3 and T3 were determined by three compartmental analyses. The PR was calculated from MCR multiplied by serum concentration. In starved rabbits, serum T4 concentrations increased from 2.1 to 3.2μg /100 ml. T4 MCR did not change and T4 PR increased significantly from 3.4 to 4.8 μg/day. Serum T3 levels did not change, but T3 MCR increased from 2.6 to 3.2 liters/day and T3 PR increased significantly from 3.5 to 4.3 μg/day. Serum rT3 concentrations increased from 3.3 to 30.8 μg/100 ml but rT3-MCR decreased significantly from 2.3 to 1.4 liters/day during starvation. Since the increase in serum rT3 levels was greater than the decrease in rT3 MCR, rT3 PR increased significantly from 78 to 416 μg/day in starved rabbits. These data indicate that in rabbits, the secretion of T4 from the thyroid glands increases and the production of T3 and rT3 (peripheral conversion of T4 and/or thyroidal secretion) increases during starvation. The increase of serum rT3 concentrations during starvation is due to both an increase in the production of rT3 and a decrease in the degradation of rT3.

Iodothyronine metabolism in liver and kidney homogenates from hyperthyroid and hypothyroid rats.

The effect of hyperthyroidism and hypothyroidism on conversion of T4 to T3 and the degradation of rT3 was studied in rat liver and kidney homogenates. Administration of T4 (5 μg/100 g BW/day for 5 days) to normal rats resulted in significant increases in the rates of both reactions in liver homogenates; the increase was no greater after T4 doses of 10 and 20 μg/100 g/day. Responses also were no greater after 12 than after 5 days of T4 treatment. After thyroidectomy, T3 production declined gradually to 30% of control in 6 weeks; rT3 degradation initially decreased more rapidly, but at 4 and 6 weeks, the percentage of fall was similar for both reactions. No significant changes in either reaction were found in kidney homogenates from T4-treated rats. In kidney homogenates from thyroidectomized rats, both reactions were significantly impaired at 6 weeks. These results extend previous observations that 5′-deiodination of T4 and rT3 change in parallel directions after various experimental manipulations, and fur...

Opposite variations in serum T3 and reverse T3 during propylthiouracil treatment of thyrotoxicosis.

Blood samples for determination of serum total and free reverse triiodothyronine (rT3), triiodothyronine (T3) and thyroxine (T4) were obtained daily in 6 previously untreated thyrotoxic patients during periods of propylthiourazil (PTU) (600 mg per day) or methimazol (MMI) (45 mg per day) administration. PTU induced about 60 per cent increase in both total and free serum rT3. This was accompanied by a rapid decrease in serum T3 and a more gradual decline in serum T4. MMI administration to untreated patients was followed by a gradual parallel decrease in rT3, T3 and T4. Turn from PTU to MMI produced a rapid decrease in serum rT3 and increase in serum T3 in all 6 patients. The relative variations in the free and total concentrations of iodothyronines were practically identical. The increase in serum rT3 after PTU is most likely explained either by enhanced deiodination of T4 to rT3 or by an inhibitory effect of PTU on rT3 degradation.