Thyroid hormone regulation of extrathyroidal iodoproteins.

The effect of thyroid status on plasma and tissue levels of labeled nonextractable iodine (NEI) derived from the metabolism of radioiodothyronines was examined in the rat. Concentrations of radioiodoprotein were substantially elevated in plasma, kidney, and liver in thyroidectomized animals 72 h postinjection of [125I]triiodothytonine ([125I]T3). Similarly, total rat concentrations of radioactive NEI were increased (52%) 72 h after injection of [125I]T3. NE125I concentrations from [125I]T3 in plasma, kidney, and liver were diminished progressively in thyroidectomized animals maintained on increasing doses of thyroxine replacement, demonstrating that iodoprotein levels were inversely related to thyroid state. The plasma disappearance rate of radioiodoprotein from [125I]T3 was markedly slowed in hypothyroid animals and accelerated in intact controls rendered hyperthyroid with daily injections of T4, 8 mug/100 g BW. Propylthiouracil (PTU) treatment of thyroidectomized rats maintained on T4, 2 mug/100 g BW per day resulted in increased NE125I from [125E]T3 in plasma, kidney, and liver. The results of the foregoing investigations suggest that thyroid hormone regulates levels of iodothyronine-derived iodoproteins by influencing the rate of degradation of iodoproteins. Moreover, the observed elevation of iodoprotein levels in T4-maintained thyroidectomized animals after PTU administration appears consistent with the modification of thyroid status due to the peripheral antithyroxine effect of PTU.     

Conversion of thyroxine to 3,5,3'-triiodothyronine in several rat tissues in vivo: the effect of hypothyroidism

The local conversion of thyroxine (T4) to 3,5,3'-triiodothyronine (T3) has been recognized as a source of T3 at various sites in euthyroid rats. The present study was designed to evaluate the effect of hypothyroidism on the source and quantity of T3 at several of these sites (liver, cerebral cortex (Cx), thymus, testis, brown adipose tissue). For this purpose intact euthyroid rats and radiothyroidectomized (RTx) rats received a continuous iv infusion of [125I]T4 and [131I]T3 until isotopic equilibrium was attained. In addition to the labelled iodothyronines, RTx rats received a continuous iv infusion of 0.75 microgram T4/day, in order to maintain a defined hypothyroid state. At the end of the infusion period the animals were bled and perfused, and homogenates of the various organs were prepared. The mean plasma T4 and T3 levels in T4-maintained RTx rats, as measured by RIA, were 1.5 micrograms/dl and 15 ng/dl (euthyroid values: 5.2 micrograms/dl and 48 ng/dl, respectively). The plasma and tissue homogenates were processed for thin layer chromatography and the [125I]T4, [125I]T3 and [131I]T3 levels determined. From these data the concentrations of T4, total T3 and T3 derived from local T4 to T3 conversion (LcT3(T4)) in tissue could be calculated. The relative mean contribution of LcT3(T4) to the total T3 in Cx (75%), thymus (31%), testis (43%) and brown adipose tissue (65%) from hypothyroid rats was higher than that determined for euthyroid animals (66%, 19%, 29% and 27%, respectively). The reverse was found for the liver (15% vs 39%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentrations of thyroxine and 3,5,3'-triiodothyronine at 34 different sites in euthyroid rats as determined by an isotopic equilibrium technique

The present study was designed to assess the quantities of T4 and T3, and the source (i.e. plasma-derived vs. locally produced) of the latter iodothyronine, in various rat tissues. For this purpose, normal intact rats were brought to isotopic equilibrium by means of a continuous iv infusion of [125I]T4 and [131I]T3 for a prolonged period. At the end of the infusion period, the animals were bled and perfused. Either whole small organs or weighed portions of tissues were homogenized in saline. The iodothyronines were extracted with ethanol-ammonia and separated by TLC. The [125I]T3/[131I]T3 ratios for the tissue homogenates and plasma were determined, and the relative contribution of the T3 derived from local T4 to T3 conversion [abbreviated: Lc T3 (T4)] to the total T3 in a given tissue was calculated. The endogenous T4 and T3 levels in the various organs were computed from the known specific activities of the labeled iodothyronines. The concentration of T4 in plasma greatly exceeded that found for tissue. Among the tissues examined, the T4 concentration was highest in the liver and lowest in cerebral cortex and cerebellum. T3 (per gram) was most abundant in the kidney and anterior pituitary gland and least abundant in the testis, epididymis, and erythrocytes. In contrast to the other tissues investigated, the concentration of T3 in several regions of the brain and anterior pituitary gland either equalled or exceeded that of T4. Plasma exhibited by far the lowest T3/T4 ratio. For most of the organs investigated the contribution of Lc T3(T4) appeared to be low. On the other hand, in 15 tissues, including the central nervous system, the local production of T3 accounted for one fifth or more of the total T3 content. Although there were no regional differences between the total T3 levels in the brain, the relative contribution of Lc T3(T4) was 65% in the cerebral cortex and only 22% in the spinal cord. The variation in the source of T3 in the various parts of the central nervous system may be related to regional differences in T4 and T3 metabolism. The fact that the present study demonstrates that the relationship between circulating T3 and intracellular T3 varies from one organ to the next may be important for accurate interpretation of plasma T4 and T3 levels and for designing optimal thyroid hormone replacement therapy for patients with hypothyroidism.     

The contribution of local tissue thyroxine monodeiodination to the nuclear 3,5,3'-triiodothyronine in pituitary, liver, and kidney of euthyroid rats.

The contributions of local T4 monodeiodination and plasma T3 to the nuclear T3 of anterior pituitary, liver, and kidney were measured in euthyroid rats. After injection of [125I]T4, there was a gradual increase in the quantity of plasma [125I]T3 in excess of injected contaminant, which peaked at approximately 12 h after injection and remained a constant fraction of plasma [125I]T4 (2.8 X 10(-3) after that time. In the nuclei of anterior pituitary tissue, there was also a slow increase in locally produced [125I]T3 (in excess of that which could be accounted for by plasma [125I]T3) which appeared to peak at about 16 h after [125I]T4 administration. The ratio of nuclear [125I]T3 generated intracellularly to plasma [125I]T4 was constant at 18 and 24 h after T4 injection and was 13 +/- 2 X 10(-3) in nuclei of pituitary, 2.0 +/- 0.4 x 10(-3) in liver, and 0.47 +/- 0.1 x 10(-3) in kidney (all values are mean +/- SD). This locally generated T3 resulted in a markedly higher nuclear to plasma (N:P) ratio for [125I]T3 than for injected [131I]T3 in the same animals. The N:P ratio for [125I]T3 at equilibrium after injected T4 was 2.4 +/- 0.6, 0.47 +/- 0.09, and 0.10 +/- 0.03 (nanograms of T3 (mg DNA)-1/ng T3 ml-1) in pituitary, liver, and kidney. Comparable values for [131I]T3 N:P ratios were 0.47 +/- 0.14 (pituitary), 0.18 +/- 0.01 (liver), and 0.036 +/- 0.008 (kidney). Using RIA values for plasma T4 and T3 concentrations in these rats and maximal nuclear T3-binding capacities estimated in parallel experiments, the gravimetric quantities of nuclear T3 derived from plasma T3 and from local T4 to T3 monodeiodination were estimated and expressed as the percentage of saturation of T3 receptors. Seventy-eight percent of nuclear T3 receptor sites in anterior pituitary were occupied with one-half of the nuclear T3 derived directly from plasma T3 and the other half from intrapituitary T4 monodeiodination. Local T4 monodeiodination provided only 28% and 14%, respectively, of the nuclear T3 in liver and kidney, and the nuclear receptors of these tissues were about 50% saturated. Since our previous studies have shown that the occupancy of the pituitary nuclear T3 receptors may regulate TSH release, these data provide a mechanism by which TSH secretion could be altered by changes in either plasma T3 or T4, whereas nuclear T3 in liver and kidney is predominantly a function of the plasma T3 concentration.     

Targeted disruption of the type 1 selenodeiodinase gene (Dio1) results in marked changes in thyroid hormone economy in mice

The type 1 deiodinase (D1) is thought to be an important source of T3 in the euthyroid state. To explore the role of the D1 in thyroid hormone economy, a D1-deficient mouse (D1KO) was made by targeted disruption of the Dio1 gene. The general health and reproductive capacity of the D1KO mouse were seemingly unimpaired. In serum, levels of T4 and rT3 were elevated, whereas those of TSH and T3 were unchanged, as were several indices of peripheral thyroid status. It thus appears that the D1 is not essential for the maintenance of a normal serum T3 level in euthyroid mice. However, D1 deficiency resulted in marked changes in the metabolism and excretion of iodothyronines. Fecal excretion of endogenous iodothyronines was greatly increased. Furthermore, when compared with both wild-type and D2-deficient mice, fecal excretion of [125I]iodothyronines was greatly increased in D1KO mice during the 48 h after injection of [125I]T4 or [125I]T3, whereas urinary excretion of [125I]iodide was markedly diminished. From these data it was estimated that a majority of the iodide generated by the D1 was derived from substrates other than T4. Treatment with T3 resulted in a significantly higher serum T3 level and a greater degree of hyperthyroidism in D1KO mice than in wild-type mice. We conclude that, although the D1 is of questionable importance to the wellbeing of the euthyroid mouse, it may play a major role in limiting the detrimental effects of conditions that alter normal thyroid function, including hyperthyroidism and iodine deficiency.     

Binding of endogenous iodothyronines to isolated liver cell nuclei

The metabolic role of a number of the metabolites of T4 is unknown. Hence, these iodothyronines, now known to be present in human serum, were tested for their ability to displace [125I]T3 from specific binding sites in isolated pig liver nuclei. Compared with T3 (1.0), the molar inhibition ratios of the analogs tested were: triiodothyroacetic acid, 4.4; T4 6.2; 3.3'-diiodothyronine, 56; 3,5-diiodothyronine, 245; rT3, 264; and 3',5'-diiodothyronine, 60,000. In isolated pig liver nuclei, the Ka for T3 was 1.73 +/- 0.21 X 10(9) M-1 and that for T4 was 0.17 +/- 0.06 X 10(9) M-1. Nuclei stored in liquid nitrogen for up to 8 weeks leaked bound [125I]T3 into the supernatant during the incubation period. No loss of bound [125I]T3 was observed with freshly prepared nuclei. The data indicate that, with the exception of T3 and T4, iodothyronines derived from T4 are unlikely to modulate the interaction of T3 with its receptor unless their perireceptor concentration is significantly greater than their serum concentration.     

The influence of partial food deprivation on the quantity and source of triiodothyronine in several tissues of athyreotic thyroxine-maintained rats

In the present study the influence of partial food deprivation (PFD) on the quantity and source of T3 [i.e. T3 derived from local T4 to T3 conversion (Lc T3 (T4] vs. plasma-derived T3] in several rat tissues was investigated. Two groups of athyroid rats on a synthetic diet received a continuous iv infusion consisting of T4 (1.0 microgram/100 g BW X day), [125I]T4, and [131I]T3 over a prolonged period. For one group of rats the daily food intake was restricted by one third to maintain constant BW during the infusion period. At isotopic equilibrium the mean plasma T3, T4, and TSH levels for control-fed rats were: 38 ng/dl, 5.1 micrograms/dl, and 470 ng/ml, respectively. The values for rats on PFD were T3: 22 ng/dl, T4: 4.8 micrograms/dl, TSH: less than 70 ng/ml. The [125I]T3 and [131I]T3 contents of whole homogenates from liver, kidney, thigh muscle, cerebral cortex, cerebellum, and anterior pituitary gland as well as the subcellular fractions from liver, kidney (nuclei, mitochondria, microsomes, and cytosol), and anterior pituitary gland (nuclei) were determined (after extraction in ethanol) by thin layer chromatography. The contribution of Lc T3(T4) and the total T3 levels in these tissue preparations could then be calculated. In the cerebral cortex, cerebellum, and anterior pituitary gland of PFD rats plasma-derived T3 as well as Lc T3(T4) was decreased. The total T3 level in the liver did not change under PFD, owing to an increase in Lc T3(T4). It is possible that the release of hepatic Lc T3(T4) into the blood stream was reduced. In neither group was there appreciable Lc T3(T4) in muscle. In contrast to the other tissues investigated, the [131I]T3 tissue-plasma ratio for muscle had increased under PFD, suggesting a higher uptake of T3. As a consequence, the T3 levels in the muscles of PFD rats did not differ from those in normally fed animals. For both groups of rats the contribution of Lc T3(T4) in hepatic nuclei was far lower than that found for the other hepatic cellular fractions. This would suggest that the hepatic nucleus preferentially takes up plasma-derived T3. In both control-fed and PFD rats the bulk of renal T3 appeared to be exchangeable with plasma T3. Hence the T3 levels in renal nuclei were reduced under PFD. The nuclear T3 levels in the anterior pituitaries from PFD rats were markedly decreased. Therefore it is likely that other factors determine TSH secretion under PFD.     

Conversion of thyroxine to 3,3',5'-triiodothyronine in the guinea pig placenta: in vivo studies

Studies of placental inner-ring deiodination of T4 were carried out in pregnant guinea pigs, by in situ placental perfusion. When [131I]T4 and [125I]rT3 were administered to the mother, the ratio of fetal side to maternal side [131I]rT3 was more than 10 times greater than the corresponding ratio for [125I]rT3. When radiolabeled T4 was supplied to the fetal side of the placenta in perfusion fluid, and the perfusate recycled through the placental circuit, there was a progressive increase in labeled rT3 concentration in the perfusate. These results indicate that the guinea pig placenta actively deiodinates both maternal and fetal T4 in the inner ring in vivo. We found evidence of very little outer ring deiodination of either T4 or rT3. The quantitative contribution of placental deiodination of maternal T4 to circulating rT3 in the fetus appears to be small; however, placental deiodination of fetal T4 (about 0.5 nmol/kg fetal BW X day) could contribute significantly to fetal rT3 levels. Our observations are consistent with the hypothesis that placental inner-ring deiodination of T4 plays a part in the regulation of fetal iodothyronine metabolism.     

The turnover of thyroxine in the plasma of the domestic fowl (Gallus domesticus)

A method for estimating thyroid activity in the chicken by measuring the plasma[¹²⁵I]-thyroxine ([¹²⁵I]T₄) degradation rate is described. The initial disappearance curve for plasma [¹²⁵I]T₄ was triphasic representing the dilution, extravascular distribution, and fractional turnover of T₄, respectively. The curve represented fractional turnover between 2 and 9 hr after [¹²⁵I]T₄ injection. Thereafter, it changed as the proportion of tri-[¹²⁵I]iodothyronine increased and radioiodide was recycled. The plasma half-life of thyroxine was 206 min when total plasma ¹²⁵I was used as an estimate of [¹²⁵I]T₄ but 138 min when the hormonal fraction was extracted on cation-exchange resin. Plasma protein-bound radioiodide overestimated [¹²⁵I]T₄ due to the presence of as yet unidentified nonhormonal iodinated proteins.     

Comparative aspects of the distribution, metabolism, and excretion of six iodothyronines in the rat

We have studied the kinetics of 3 iodothyronines, 3,3'-diiodothyronine (T2), 3',5'-T2, and 3'-monoiodothyronine (T1), in groups of young adult male rats maintained under normal steady state physiological conditions. We have also performed a comparative analysis of these results, combined with corresponding kinetic indices of T4, T3, and rT3, to obtain a more comprehensive understanding of normal thyroid hormone production, distribution, and metabolism. Tracer doses of 125I-labeled 3,3'-T2, 3',5'-T2, and 3'-T1 were separately injected iv, and blood samples were collected 6-12 times for each iodothyronine in optimized sequential kinetic studies designed to maximize the precision of kinetic parameters. Labeled iodothyronines were separated quantitatively from their metabolites in each plasma sample by Sephadex G-25 column chromatography. Conventional kinetic analysis of the resulting data generated distribution volume, clearance, turnover, and mean residence time indices for each iodothyronine, and concomitant compartmental analysis of the same data provided additional results useful for integration and comparative analysis of the 6 iodothyronines. Kinetic parameters for all but T4 and T3 were similar, suggesting that similar mechanisms are responsible for the transport, metabolism, and distribution of nonhormonal iodothyronines. All but T4 and T3 (and, to a much lesser extent, 3'-T1) were almost completely and irreversibly metabolized, whereas 24-30% of the hormones (and 6% of 3'-T1) were excreted as such in feces only. Three-pool models fitted individual plasma kinetic data sets best in all cases (for all 6 iodothyronines), each with a plasma, a slowly exchanging (slow), and a rapidly exchanging (fast) pool, and kinetic parameters of interest were quantified for each iodothyronine (Ti). Quantitative analysis of an integrated 18-pool model for all 6 Tis revealed several other features of physiological interest. The fractional transport rate of T3 into the fast pool (liver, at least) is about an order of magnitude larger than that for all other Tis, supporting the hypothesis that transport of T3 into fast tissues (e.g. liver cells) is selectively amplified relative to that of the 5 other iodothyronines studied. Simultaneous and direct comparison of the 6 plasma kinetic data sets also supports this result. In addition, composite slow tissue pools, which probably exclude liver and kidney, contained the largest whole body fractions of all Tis (greater than 50%), and these also appear to be major sites of whole body T4 monodeiodinations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Brain cortex reverse triiodothyronine (rT3) and triiodothyronine concentrations under steady state infusions of thyroxine and rT3

T4 and reverse T3 (rT3) can inhibit 5'-deiodinase type II activity in rat brain cortex, pituitary, and brown adipose tissue, raising the possibility that T4 may act in vivo after conversion to rT3. The aim of this study was to measure in hypothyroid (Tx) rats the content of brain cortex rT3 during a constant 7-day infusion of either [125I]T4 alone, corresponding to 12 pmol T4/day X 100 g body weight (BW), or together with 400 pmol T4/day. [125I]T4, rT3, and T3 were extracted from brain cortex, pituitary, kidney, and liver with a combination of adsorption chromatography on Sephadex G-25, HPLC, and immunoprecipitation. [131I]T4, T3, or rT3 were used as internal standards. [125I]rT3 could be detected in brain cortex, liver, and kidney in Tx rats infused with [125I]T4 (12 pmol T4/day X 100 g BW) and in those infused with 400 pmol T4/day X 100 g BW. The highest rT3 concentrations were found in brain cortex, where it represented 6% to 10.5% of the local T4 concentration. During an infusion of 400 pmol T4/day X 100 g BW, brain cortex T3 concentration was 6 times higher in the brain cortex than in serum, and even exceeded that of T4. In Tx rats receiving [125I]T4 alone the brain cortex to serum T3 ratio was 3:1, but the total serum T3 concentration, measured by RIA, was much higher than that due to conversion [0.50 +/- (SE) 0.1 pmol/ml vs. 0.018 +/- 0.002 pmol T3/ml], indicating thyroidal secretion. The effect of the blood-brain barrier on rT3 was measured by infusing [125I]rT3 over 4 days. After killing, rT3 was isolated as above. Approximately 3% of serum rT3 was retrieved from the brain cortex, whereas during the T4 infusion 40-50% of serum rT3 was found demonstrating that brain cortex rT3 is locally produced.     

Effects of human thyroxine-binding globulin and prealbumin on the reverse flow of thyroid hormones from extravascular space into the bloodstream in rabbits

A plasma fraction rich in thyroid hormone-binding globulin (hTBG, human thyropexin) was injected iv into rabbits in order to see whether thyroid hormone concentrations in plasma would increase by return of T3 and T4 from the extravascular space. For this purpose, both [125I]T3 and [131I]T4 were simultaneously injected. After 1 h, or after 16 h in another series of experiments, 50 mg hTBG were injected iv. Thereafter, the mean radioactivity of both [125I]T3 and [131I]T4 in the plasma rose, and reached its peak 20-30 min after hTBG injection; [125I]T3 and [131I]T4 returned to the preinjection value slowly, after more than 3 h. When hTBG was injected 15-16 h after the radioactive hormones, the mean radioactivity of [125I]T3 reached its peak about 1 h after hTBG injection and returned to the base value after approximately 5.5 h, [131I]T4 reached its peak about 1 h after hTBG injection and returned to the base value within 12 h. After injection of hTBG, total T4 and T3 concentrations in plasma increased about 3- to 5-fold over the base values. At the same time, the percentage of both, free T4 and free T3 dropped instantly whereas absolute free T4 and free T3 values remained almost constant. After injection of 500 mg transthyretin (hTBPA), a similar flux of [125I]T3 and [131I]T4 was observed, whereas 500 mg human serum albumin were ineffective. These marked effects of injected hTBG and hTBPA on the serum levels of [125I]T3, [131I]T4, and total T3 indicate that reentry of T3 and T4 into the intravascular compartment is an important component of thyroid hormone distribution and transport. As can be anticipated from the animal experiments, the efficiency of plasmapheresis or hemofiltration methods may be improved by previous application of large doses of hTBPA or hTBG in cases of thyrotoxicosis.     

Effects of 5,5'-diphenylhydantoin on thyroxine and 3,5,3'-triiodothyronine concentrations in several tissues of the rat

We studied the effect of 5,5'-diphenylhydantoin (phenytoin, DPH) on the metabolism of thyroid hormones, the intracellular concentration of T4, and the source and concentration of T3. Two groups of six male Wistar rats received a continuous infusion of 10 ml saline/rat. day. One group received DPH in their food (50 mg/kg BW) for 20 days. For both groups [125I]T4 and [131I]T3 were added to the infusion fluid for the last 10 and 7 days, respectively. At isotopic equilibrium the rats were bled and perfused. Compared to the controls, plasma T4 and T3 in the DPH group were reduced (22% and 31%, respectively); TSH did not change. The rate of production of T4 and the plasma appearance rate for T3 were decreased. Thyroidal T3 production was markedly reduced. From the increased [125I]T3/[125I]T4 ratio for plasma, it follows that total body conversion was enhanced. The tissue T4 concentrations decreased in parallel with the plasma T4 level. Total T3 was reduced in all organs. In tissues in which local conversion does not occur, i.e. heart and muscle, the decrease reflected the decrease in plasma T3. In the liver both plasma-derived T3 and locally produced T3 were diminished. In cerebellum and brain the plasma-derived T3 pool was even smaller than was expected from the decrease in plasma T3. This was partly compensated by an increase in local conversion. Only for these two organs was the decrease in the tissue/plasma ratio for [131I]T3 significant. Our results suggest tissue hypothyroidism, caused by a decrease in the production of T4 and T3, which is partly compensated by increased conversion in several organs. The transport of T3 into cerebellum and brain is disturbed, which can be attributed to the mode of action of DPH.     

Characterization of human iodothyronine sulfotransferases

Sulfation is an important pathway of thyroid hormone metabolism that facilitates the degradation of the hormone by the type I iodothyronine deiodinase, but little is known about which human sulfotransferase isoenzymes are involved. We have investigated the sulfation of the prohormone T4, the active hormone T3, and the metabolites rT3 and 3,3'-diiodothyronine (3,3'-T2) by human liver and kidney cytosol as well as by recombinant human SULT1A1 and SULT1A3, previously known as phenol-preferring and monoamine-preferring phenol sulfotransferase, respectively. In all cases, the substrate preference was 3,3'-T2 > rT3 > T3 > T4. The apparent Km values of 3,3'-T2 and T3 [at 50 micromol/L 3'-phosphoadenosine-5'-phosphosulfate (PAPS)] were 1.02 and 54.9 micromol/L for liver cytosol, 0.64 and 27.8 micromol/L for kidney cytosol, 0.14 and 29.1 micromol/L for SULT1A1, and 33 and 112 micromol/L for SULT1A3, respectively. The apparent Km of PAPS (at 0.1 micromol/L 3,3'-T2) was 6.0 micromol/L for liver cytosol, 9.0 micromol/L for kidney cytosol, 0.65 micromol/L for SULT1A1, and 2.7 micromol/L for SULT1A3. The sulfation of 3,3'-T2 was inhibited by the other iodothyronines in a concentration-dependent manner. The inhibition profiles of the 3,3'-T2 sulfotransferase activities of liver and kidney cytosol obtained by addition of 10 micromol/L of the various analogs were better correlated with the inhibition profile of SULT1A1 than with that of SULT1A3. These results indicate similar substrate specificities for iodothyronine sulfation by native human liver and kidney sulfotransferases and recombinant SULT1A1 and SULT1A3. Of the latter, SULT1A1 clearly shows the highest affinity for both iodothyronines and PAPS, but it remains to be established whether it is the prominent isoenzyme for sulfation of thyroid hormone in human liver and kidney.     

Metabolism of [125I]tyramine cellobiose-labeled low density lipoproteins in squirrel monkeys

Low density lipoproteins labeled with [125I]tyramine cellobiose ([125I]TC-LDL) were removed from the circulation of squirrel monkeys at a similar but slightly slower rate than LDLs labeled with 125I, [125I]hydroxyphenyl propionic acid, or [3H]leucine. After the simultaneous injection of [125I]TC-LDL and [131I]LDL labeled with 131ICl, the 125I was also removed at a slightly slower rate than 131I. Most of the radioactivity was retained in tissues and not excreted during the 24 h after injection of [125I]TC-LDL. This finding supports the claim of Pittman et al. [18] that [125I]TC-LDL can be used to determine the irreversible uptake of LDL by different tissues. The liver cleared more LDL than any other organ, but the adrenals and ovaries were more active per gram. Trichloroacetic acid (TCA) precipitated more than 80% of the radioactivity in the tissues that had low 125I uptake, but only about 50% of the 125I in more active tissues (liver, adrenals, ovaries, and spleen). Only a small percentage of 125I in urine and bile was TCA-precipitable. In the dual label experiment with [125I]TC-LDL and [131I]LDL there was a selective retention of 125I in samples from liver, spleen, adrenals, and, perhaps testes, and an almost complete selectivity for 125I in bile and feces. The aortic intima plus inner media (AIM) cleared much less LDL than other tissues, but the uptake by the entire AIM was proportional to the cholesterol concentration and weight of the total AIM. There was, however, no correlation between either of the latter two measurements and the uptake of LDL per gram of AIM. The concentration of LDL apolipoprotein in the AIM determined by immunoelectrophoresis did not correlate significantly with LDL uptake per gram. Both the amounts of LDL apolipoprotein present and labeled LDL taken up by the AIM depended on the weight of the sample, and perhaps on the weight of intima in the sample.     

Photoaffinity labeling of rat type I iodothyronine deiodinase

The photoreactive compound p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PAL) was coupled to [125I]rT3, T4, or T3 and incubated with liver and kidney microsomes of hypo-, hyper-, or euthyroid rats to identify the type I iodothyronine deiodinase. Various substrates or inhibitors of the enzyme, including rT3, T4, T3, 6-n-propylthiouracil (PTU), and iopanoic acid, were used as competitors to establish the specificity of protein labeling. The PAL derivatization enhanced the behavior of T4 and T3 as substrates for the type I enzyme. No specific labeling of microsomal proteins was observed with either rT3 or T4-PAL, presumably due to deiodination of the labeled compound. In contrast, T3-PAL labeled a 27-kDa band, the presence of which paralleled thyroid status. The labeling of only this protein was blocked by either substrates or enzyme inhibitors in a dose-dependent fashion, with a rank order of potency predicted by the activity of such compounds in type I enzyme assays. The specific nature of these competitions provides further evidence that this 27-kDa protein, identified in previous studies using N-bromoacetyl [125I]T3 or -T4, contains the active site of the rat type I deiodinase. This is in agreement with the mol wt of the rat type I deiodinase deduced from the recently identified cDNA coding for this protein.     

Characterization and modulation of growth hormone and prolactin binding in mouse liver.

The specific binding of 125I-labeled insulin, human hormone ([125I]hGH), bovine growth hormone ([125I]bGH), and ovine prolactin ([125I]oPRL) was studied in mouse liver membranes. [125I]hGH and [125I]oPRL bound to adult liver membranes. Pregnancy increased the specific binding of [125I]hGH but not that of [125I]oPRL. [125I]hGH was displaced from membranes of pregnant mice by hGH, oPRL, and bGH, but only by hGH and oPRL from liver membranes of nonpregnant mice. Significant specific binding of [125I]bGH was seen only in pregnancy. The binding of [125I]bGH to pregnant mouse liver membranes increased with increasing concentration of either membrane protein or [125I]bGH. Both the specific binding and dissociation of [125I]bGH were greatly influenced by the time and temperature of incubation. Binding of [125I]bGH was inhibited by growth hormones, including hGH and rat GH, and not by lactogenic hormones (various prolactins and human placental lactogen), ACTH, glucagon, or insulin. The inhibition of [125I]hGH binding by hGH and bGH, in the presence of excess (2 mug/ml) of PRL, was very similar to that seen with [125I]bGH. Scatchard plots of displacement dose-response curves obtained under steady state conditions of 4C were nonlinear and very similar with either [125I]bGH or [125I]hGH. This contrasted with the linear Scatchard plots obtained from displacement dose-response curves of either [125I]oPRL or [125I]hGH in the presence of excess (2 mug/ml) bGH. Termination of pregnancy, either naturally or by hysterectomy, reduced [125I]bGH specific binding to nonpregnant levels by 24 to 36 h. Estrogen administration did not increase [125I]bGH binding in hepatic membranes. Nonpregnant mice possess hepatic lactogen binding sites which are uninfluenced by pregnancy. GH specific binding sites are markedly augmented during pregnancy. The close correlation between the level of these sites and pregnancy suggests that they are regulated by a product of the fetoplacental unit.     

The effect of free radicals on hepatic 5'-monodeiodination of thyroxine and 3,3',5'-triiodothyronine

Free radicals have been implicated in many pathological processes, including ischemia, inflammation, and malignancy. Since a reduction in extrathyroidal outer ring monodeiodination of T4 and rT3 occurs in virtually all systemic illnesses, we have studied the effect of free radicals on iodothyronine (T4 and rT3) 5'-monodeiodinating activity (MA) of liver tissue in vitro. Rat liver microsomes or homogenate were preincubated in Tris buffer for 30 min with a free radical-generating system (FRGS) and then incubated with T4 (2.5 microM) or [125I]rT3 (0.4 nM) and dithiothreitol (DTT; 5-20 mM with T4 and 20-150 mM with [125I]rT3) in the same buffer for 10 or 30 min. T3 generated during incubation was quantified by RIA of ethanol extracts of the incubation mixture. 125I generated from [125I]rT3 was quantified after precipitation of the incubation mixture with trichloroacetic acid or by paper chromatography. Free radicals caused 55% or more reduction in hepatic T4 MA and 44% or more reduction in rT3 MA in various experiments. The inhibition of hepatic rT3 MA after incubation with FRGS persisted despite removal of FRGS and washing of microsomes preincubated with FRGS before studying the MA. However, inclusion of DTT (1-60 mM) during preincubation of tissue with FRGS prevented the FRGS-induced inhibition of rT3 MA. Depletion of the iodothyronine substrate did not occur when FRGS inhibited T4 and rT3 5'-monodeiodination. Free radical scavengers, i.e. superoxide dismutase (600 IU/ml), catalase (300 U/ml), tocopherol (10 mg/ml), thiourea (0.15 M), and tert-butanol (0.15 M), all significantly reduced the inhibition of hepatic rT3 MA caused by FRGS. The FRGS-induced inhibition of hepatic T4 MA was reduced by the same doses of tocopherol, thiourea, and tert-butanol, but not by superoxide dismutase or catalase. Since free radicals may effect tissue damage by lipid peroxidation and since the latter results in generation of malondialdehyde (MDA) as a by-product of the reaction, we studied MDA by its reaction with 2-thiobarbituric acid. Incubation with FRGS caused an approximately 100-fold increase in MDA formation in liver microsomes. Serum MDA was significantly higher in 16 NTI patients than in 8 normal subjects and also higher in turpentine oil-injected rats [an experimental model of nonthyroidal illness (NTI)] than in saline-injected control rats. The data suggest that generation of free radicals may contribute to the reduced extrathyroidal 5'-monodeiodination of T4 and rT3 in NTI.(ABSTRACT TRUNCATED AT 400 WORDS)     

A theoretical five-pool model to evaluate triiodothyronine distribution and metabolism in healthy subjects.

We have examined the in vivo production, distribution, metabolism, and excretion of radiolabeled triiodothyronine (T3) in eight normal humans using a new five-pool mammillary model which includes four extravascular pools to represent liver, kidney, skeletal muscle, and all other unquantified tissues. Trace amounts of [125I]T3 and [131I]T3 were injected and plasma, stool and urine samples and external counting of liver, kidney, and thigh were drawn following bolus injection of radiolabeled T3. Our results indicate that the skeletal muscle pool represents the largest pool in our system, being 42% of the total-body pool size of the hormone (Qtot = 0.52 +/- 10% [CV] micrograms/kg body weight [BW]). The plasma pool QA (0.090 +/- 9% [CV] micrograms/kg BW) contains approximately 17% of Qtot, while the size of the unquantified tissue pool QE (0.150 +/- 16% [CV] micrograms/kg BW) is approximately 29% of Qtot. Furthermore, the size of liver pool QB and the size of kidney pool QC are about 10% and 2%, respectively, of Qtot. The rate of T3 metabolized in the skeletal muscle pool and in the unquantified composite tissue pool, as a sum, represents approximately 53% of the plasma appearance rate of the hormone (PAR3), while the rate of T3 metabolized in the liver and kidney pools represents 27% and 3% of PAR3, respectively. The rate of T3 excreted via feces and urine, as a sum, accounts for about 20% of PAR3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyroxine (T4) transport and distribution in rats treated with EMD 21388, a synthetic flavonoid that displaces T4 from transthyretin.

To test whether plasma transthyretin (TTR) might play a specific direct role in the transfer of T4 from the plasma to tissues, in vivo kinetic studies were performed in control rats and in rats treated with EMD 21388, a synthetic flavonoid that displaces T4 from TTR. The plasma disappearance curves of simultaneously injected [125I]T4 and [131I]albumin were analyzed to determine the rate constant for the transfer of T4 from the extracellular compartment to the rapidly exchangeable intracellular compartment (KE) and the steady state distribution ratio of T4 between the rapidly exchangeable intracellular compartment and the extracellular compartment (Imax/Emin). When rats were injected ip with EMD 21388 (2 mumol/100 g BW), the free T4 fraction in serum increased approximately 8-fold. This was due to displacement of T4 from TTR, as assessed by electrophoresis of serum proteins in the presence of [125I]T4. Concomitantly, both KE and Imax/Emin increased 6-fold in the treated rats. These results fail to confirm a major specific role for TTR in the transfer of T4 from the plasma to tissues. Instead, they are consistent with both the free hormone transport hypothesis and the free hormone hypothesis in this setting.