Trust your Endocrinologist - Report and Recommendations on the Ordering of Reverse T3 Testing.

Laboratory testing for markers of thyroid function is essential for the diagnosis of thyroid disease, yet, the landscape of thyroid function testing is complex and inappropriate test orders are common. Reverse T3 (rT3) is frequently seen on thyroid function testing menus as a marker of nonthyroidal illness. However, the diagnostic utility of rT3 for this indication is questionable, and testing of rT3 is not recommended by any professional practice guidelines. We reviewed a set of rT3 orders at our institution, and identified that 11 of 20 orders appeared inappropriate with respect to clinical context. These orders were less likely to have been placed at the recommendation of an endocrinologist relative to appropriate orders. We recommend that all providers refer to professional guidelines for thyroid function testing, and consult with an endocrinologist for appropriate usage of esoteric or non-standard thyroid function tests.

Rapid Identification of Unknown Organic Iodine in Small-Volume Complex Biological Samples Based on Nanospray Mass Spectrometry Coupled with in-Tube Solid Phase Microextraction.

A new method for rapid screening of unknown organic iodine (OI) in small-volume complex biological samples was developed using in-tube solid phase microextraction (SPME) nanospray mass spectrometry (MS). The method proposed a new identification scheme for OI based on nanospray high-resolution mass spectrometry (HR-MS). The mass ranges of OI ions were confirmed using the t-MS2 scan mode first; then, the possible precursor ions of OI were selected and identified orderly in full MS/ddMS2 and t-MS2 scan modes. Besides, in-tube SPME was used for the pretreatment of small-volume biological samples, and it was the first time in-tube SPME combined with nanospray MS for OI identification. The whole analysis procedure took only 8 min and consumed 50 µL per sample. Using the new method, six kinds of OI added to urine and an unknown OI C12H23O11I in human milk were successfully identified. Moreover, the proposed identification scheme is also suitable for other ambient mass spectrometry (AMS) to determine unknown compounds with characteristic fragment ions.

5'-deiodinase activity and circulating thyronines in lactating cows.

To investigate the correlation between lactation and thyroid hormone metabolism, the authors studied concentrations of total and free thyroxine (T4 and fT4), triiodothyronine (T3 and fT3), and reverse triiodothyronine (rT3) in plasma and milk, as well as liver and mammary gland 5'-deiodinase (5'D) activity in dry, early, middle, and late lactating dairy cows. Cows in early lactation show lower plasma levels of T4 and rT3 than dry, middle, and late lactating animals, whereas T3 shows the lowest plasma levels in the dry period; free T4 and T3 show a similar pattern. In early lactation there is a clear decrease in liver 5'D associated with a notable increase in mammary 5'D. Concentrations of T4 and T3 in milk drop significantly in the first few days after delivery, whereas rT3 increases up to the fourth month. The findings suggest a relationship between the hypothyroid status of lactating cows and the rearrangement of organ-specific 5'-deiodinase activity related to the maintenance of the udder's function.

Thyroid hormones in human tumoral and normal nervous tissues.

We have studied T4 and T3 concentrations, DNA and protein concentrations and 5' and 5 deiodinases in samples of brain tumors obtained at surgery from 49 patients, and, in most cases, also from surrounding normal tissue. T4 concentrations in normal cortical tissue (6.19+/-0.45 ng/g) were lower than in white matter, but the difference disappeared when referred to the DNA content (2.26+/-0.27 ng/mg DNA). No other differences were found between cortical and white matter, or among cortical lobes. T4 in normal tissue was higher than previously reported, mostly from autopsy samples, whereas T3 (0.99+/-0.07 ng/g) was similar. 5'D-I activity was negligible as compared to 5'D-II (8.11+/-1.09 fmol/h/mg protein). When expressed in relation to the different DNA contents of normal vs. tumoral tissue, 5'D-II activities were the same for both. 5D activity was highly variable in the tumoral tissue, with negligible activities in meningiomas and pituitary adenomas. When referred to the DNA content, T4 and 5'D-II were the same, but T3 concentrations were lower in the tumor (0.24+/-0.03 ng/mg DNA) as compared to normal (0.35+/-0.04 ng/mg DNA) tissue samples. Whether or not this decrease of T3 affects the expression of T3-sensitive processes remains to be studied.

Decreased reverse T3 levels in neonates with central hypothyroidism.

Concern arises when a sick infant is found to have a low serum T4, normal thyroid hormone binding, and a nonelevated thyroid-stimulating hormone. Hypothyroxinemia in this situation can result from either euthyroid sick syndrome or central hypothyroidism. To help distinguish between these diagnostic possibilities, we have measured reverse T3 and other thyroid function chemistries in six neonates who have central hypothyroidism in association with hypopituitarism. We found that these infants all had reverse T3 levels that were much lower than reported normal levels for premature and term neonates. This finding suggests that low reverse T3 levels can help to distinguish infants with central hypothyroidism from sick and well infants who tend to have relatively elevated reverse T3 levels.

Divergent deiodination of thyroid hormones in the separated parts of the fetal and maternal placenta in pigs.

Previous work from this laboratory has shown that the thyroid gland of the fetal pig begins to function at about day 46-47 (0.40-0.415 fraction of gestational age). Sera from fetuses contain lower thyroxine (T4), 3,3',5-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3) concentrations than maternal sera, except for about 2 weeks before term. The fetal T4 metabolism is dominated by the 5'-monodeiodinating activity (5'-MD). In the present study we measured the iodothyronines content, and the outer (5'-MD) and inner (5-MD) monodeiodinases activity, in homogenates of the placenta. The pig placenta, which is of the epitheliochorial type, was separated into the fetal and the maternal part. The concentrations of T4, T3 and rT3 were lower, and the deiodinating activity of 5'-MD and 5-MD higher, in the fetal than in the maternal placenta. The fetal placenta not only deiodinated more actively T4 to T3 and T4 to rT3, but degraded T3 to 3,3'-diiodothyronine (3,3'-T2) more actively than rT3 to 3,3'-T2. Such divergent deiodinating activity of T4 to T3, T3 to 3,3'-T2 and rT3 to 3,3'-T2 might favor establishing a relatively high and constant rT3 concentrations in fetal and maternal placentas, and a lower T3 in the fetal placenta. The inner ring deiodinating activity (excluding a day before parturition) was always more active in the fetal placenta, while the outer ring deiodinations varied in this respect, depending on the gestation stage. These results support the hypothesis that in the fetal pig, enzymatic deiodination of thyroid hormones forms a barrier which reduces transplacental passage of the hormones and that the fetal part of the placenta is the primary factor in the mechanism regulating the hormonal transfer. In spite of the presence of the barrier, there is an adequate maternal supply of thyroid hormones to the fetus in early gestation, which suggests that the enzymatic mechanism is influenced in some way by the thyroid status of the fetus.

Amniotic and allantoic fluid concentrations of thyroxine, 3,3',5'-tri-iodothyronine, 3,3'-di-iodothyronine and 3',5'-di-iodothyronine in the pig during the period of gestation.

Thyroxine (T4), 3,3',5'-tri-iodothyronine (reverse T3; rT3) and di-iodothyronines (3,3'-T2 and 3',5'-T2) were measured in pig amniotic fluid (AF) and allantoic fluid (Al) between 32 and 113 days of normal pregnancy. Low but measurable quantities of T4 in AF and Al (2.1 +/- 0.3 and 3.2 +/- 0.5 nmol/l respectively) were found before the onset of fetal thyroid gland function, which indicates the maternal source of T4. The presence of rT3 (55.8 +/- 4.1 pmol/l in AF and 49.8 +/- 5.3 pmol/l in Al), 3,3'-T2 (45.5 +/- 0.6 pmol/l in AF and 49.2 +/- 9.2 pmol/l in Al) and 3',5'-T2 (20.8 +/- 2.6 pmol/l in AF and 24.0 +/- 2.2 pmol/l in Al) may be attributed to the monodeiodinase system already active in fetal pig tissues in early pregnancy, as demonstrated previously. T3 concentration was undetectable in both AF and Al. An approximately twofold increase in the levels of T4, rT3 and T2s in AF and Al at mid-gestation was observed. T4 and rT3 in AF showed a positive correlation with protein concentrations. AF rT3 concentration (but not T4) correlated with rT3 in the cord and maternal serum. The 3,3'-T2 and 3',5'-T2 in AF and Al showed parallel changes to rT3, while the rT3/3,3'-T2 and rT3/3',5'-T2 molar ratios remained constant. T4 concentrations in AF and Al were markedly lower than in corresponding maternal and fetal serum; the rT3 concentration in Al was equal to that in AF and two to four times lower than in fetal serum. In spite of differences between serum hormone patterns in the pig and human near term, iodothyronine concentrations in AF showed some similarities, mainly the following: undetectable T3, a strong correlation between rT3, T4 and AF total protein and the presence of 3,3'-T2 and 3',5'-T2 in measurable levels. Comparative data for Al, except the ones in the present study in the pig, are not available.

Differences in the thyroxine, triiodothyronine and reverse triiodothyronine contents of fetal pig tissues relative to gestational age.

The concentrations of thyroxine (T4), 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (reverse T3) were measured in the liver, kidney and skeletal muscles of pig fetuses from day 32 to 113 of age. The iodothyronines were found to be present already between days 32 and 39, that is before the onset of fetal thyroid function. In the liver, kidneys and skeletal muscles T4 reached maximal concentrations at midgestation (days 56 and 93). The T3 level increased with gestational age in all tissues examined but, as opposed to T4, its maximal value was reached shortly before birth. The rT3 level was the highest between days 85 and 100 in the liver and kidney; however, in the skeletal muscles it did not change with gestation. The presented profiles of change of T4 and rT3 in the examined tissues did not correspond to the blood hormone pattern already observed in pig, whereas changes in tissue T3 concentrations were similar to those in the serum.

Immediate and dose-response related increase of biliary excretion of reverse triiodothyronine after propylthiouracil administration.

In a group of rats infused with L-thyroxine (0.13 micrograms T4 in 0.6 ml alkaline saline per hour) for 6 h to which an infusion of propylthiouracil (PTU) was added beginning from the 3rd hour (2 mg PTU in 0.6 ml saline per hour) a significant increase of biliary excretion of reverse triiodothyronine (rT3) was found. In another experiment a dose-response related rT3 excretion by bile was observed in groups of rats infused with 0.05, 0.10, 0.20 or 0.40 mg PTU in 1.2 ml alkaline saline per 2 h, all animals receiving a pulse dose of 1 micrograms rT3 at the beginning of PTU infusion. It was concluded that the increase of rT3 excretion results from the inhibition of 5'-deiodinase type I activity in the liver caused by PTU. It thus appears that such phenomenon may be used as in vivo marker of that enzyme activity.

Presence of iodothyronines in the yolk of the hen's egg.

Thyroxine (T4), 3,5,3'-triiodothyronine (T3) and 3,3',5-triiodothyronine (rT3) were determined in the yolk and white of the hen's egg and in the oocytes at various stages of development. For this purpose we have utilized the property of 0.08 N NaOH both to dilute the yolk or white and to extract and bind iodothyronines by strong alkaline Sephadex G-25. No iodothyronines were detected in the egg white. The total levels of T4 and T3/per 100 mg of yolk increased gradually with increased weight of oocytes weighing less than 6 g. Above this weight, levels of both iodothyronines were stable. The concentrations of T4 and T3 ranged from 0.6 to 1.0 ng and 150 to 230 pg/100 mg of yolk, respectively. The concentration of rT3 per 100 mg of yolk was independent of the weight of the oocytes and ranged between 10 and 100 pg/100 mg. The serum:yolk ratio oscillated between 2.62 and 1.15; 1.16 and 0.48; 0.11 and 0.15 in smallest and largest oocytes for T4, T3 and rT3, respectively. The perfusion experiment of the ovary indicates that all three iodothyronines can enter the ovary. The accumulation of iodothyronines by the ovary after 3 hr of incubation was as follows (in percentage of total radioactivity): T4, 35.85 +/- 2.94; T3, 26.73 +/- 2.97; rT3, 27.02 +/- 4.38. The highest accumulation of 125I-labelled iodothyronines per unit mass was seen in the oocytes of lowest size. The 3' or 5' deiodination of iodothyronines by the ovary, measured in the medium after 3 hr of perfusion, was negligible.

Concentration of thyroid hormones and prolactin in dairy cattle serum and milk at three stages of lactation.

Eighteen lactating Holstein cows were used with six each in early, mid, and late lactation. Blood samples were obtained on 7 successive d. Blood serum and milk were measured by radioimmunoassay for thyroxine, 3,5,3'-triiodothyronine, and 3,3',5'-triiodothyronine. Prolactin was also measured in serum by radioimmunoassay. Serum thyroxine increased as lactation progressed and milk production declined (50, 55, and 62 ng/ml). Serum concentrations of triiodothyronine and reverse triiodothyronine were unchanged throughout lactation. Prolactin in serum declined as lactation advanced linearly (14.4, 11.8, and 10.5 ng/ml). Concentrations of thyroxine and triiodothyronine in milk declined significantly between early and mid but not mid and late lactation. Reverse triiodothyronine in milk did not change over the lactation. Serum triiodothyronine contained 1200 to 1300 pg/ml, whereas that in milk was 200 to 300 pg/ml. Reverse triiodothyronine was over 300 pg/ml in serum and only 80 to 90 pg/ml in milk. Amounts of thyroxine and triiodothyronine available to offspring from milk were calculated to be minor sources (4 to 5%) of total requirements for maintenance of metabolic function.

Simultaneous observations of iodothyronine content in the thyroid gland, serum and thyroxine 5'- and 5-monodeiodinase activity in liver, during the neonatal period of the pig.

Serum T4 and rT3 were high at about 4-12 h after birth, then they decreased to a nadir on day 3 (rT3) and day 7 (T4). Serum T3 concentration fell immediately after birth but then increased to a relatively stable level during the next 2-6 weeks, then fell after weaning. Reciprocal concentration profiles of T4, T3 and rT3 in the thyroid were found. The thyroidal iodothyronine content increased significantly after weaning. In the liver, 5'-monodeiodinating activity, low after birth, rose until day 3 and then decreased concomitantly with T3 in serum. The 5-monodeiodinating activity, high at birth, fell to a nadir at about 3 weeks. No changes in 5- and 5'-deiodinase activity after 3 weeks were observed. Opposite to the variations in absolute content, the iodothyronine relative proportion in thyroid tissue was practically unchanged until weaning time (6 weeks), when they rose. Serum T3/T4 and rT3/T4 ratios increased with age until weaning. The post-weaned pigs had T3/T4 and rT3/T4 ratios about two times smaller than 6-weeks-old pigs. Serum rT3/T3, high after birth, decreased with age. Summarizing, the results indicate that neither changes in the thyroid iodothyronine content nor in the liver T4-monodeiodinating activity can solely account for variations in serum TH during the early neonatal period in the pig. It is suggested that the rapid variations in serum TH levels can reflect changes in the thyroidal secretory activity in preferential T3 secretion and/or blood disappearance rates.

Effect of changes in thyroid state on metabolism of thyroxine by rat placenta.

We studied the effect of the state of the thyroid on T4 monodeiodination in the rat placenta, and it was compared with those in the liver and kidney. The tissues, maternal serum, and amniotic fluid were obtained from pregnant rats. The tissues were homogenized in cold 50 mM Tris-HCl buffer, pH 7.5. The homogenate (1 mg protein) was incubated at 37 degrees C for 60 min with 1 microgram T4 in the presence of 5 mM DTT. The T3 and reverse T3 generated in the reaction mixture were extracted into cold ethanol and measured by RIAs. The conversion of T4 to reverse T3 in rat placenta was not significantly changed in MMI-induced hypothyroidism or T4 induced hyperthyroidism. On the other hand, conversion of T4 to T3 in the liver and kidney were changed in parallel with the thyroid state. The concentration of reverse T3 in the amniotic fluid was increased in accordance with the increase in the maternal serum T4 concentration. These results indicate that the placental T4 inner ring deiodination is not affected by the thyroid state, and that the change in the amniotic fluid reverse T3 concentration in this study is mainly dependent upon the change in maternal thyroid function.

[Amniotic fluid 3,3',5'-triiodothyronine (reverse T3)].

RT3(3,3',5'-triiodothyronine) levels in amniotic fluid and T4(thyroxine), T3(triiodothyronine), rT3 and TSH(thyroid-stimulating hormone) levels in maternal and cord serum were determined simultaneously by RIA. We also determined the activities of the monodeiodination of thyroxine to rT3 in placentas. Amniotic fluid rT3 and cord serum rT3 levels decreased, but T4, T3 and TSH levels increased with advancing gestational age. The activities of the monodeiodination in placentas decreased rapidly from midgestation, preterm to term. In maternal hyperthyroidism, amniotic fluid rT3 levels were markedly elevated. Moreover, there were significant positive correlations between amniotic fluid rT3 and maternal serum rT3 (r = 0.756, p less than 0.001, n = 26) and T4(r = 0.509, p less than 0.01, n = 26) in the normal 3rd trimester. We found significant correlations between amniotic fluid rT3 and fetal thyroid function as well as the activity of the monodeiodination in placenta after 17 weeks' gestation. But we couldn't find any such correlations in the 3rd trimester. These data suggest that the amniotic fluid rT3 in the 3rd trimester was affected by maternal thyroid function as well as fetal thyroid function and the activity of the monodeiodination in placenta.

Plasma kinetics, tissue distribution, and cerebrocortical sources of reverse triiodothyronine in the rat.

Studies in vitro have shown that rT3 is a potent and competitive inhibitor of T4 5'-deiodination (5'D). Recent studies in vivo have shown that cerebrocortical (Cx) T4 5'D-type II (5'D-II) activity [propylthiouracil (PTU) insensitive pathway], is reduced by T4 and rT3, the latter being more potent than T3 in Cx 5'D-II suppression. Some other reports had described rT3 production in rat brain as a very active pathway of thyroid hormone metabolism. To examine the possibility that rT3 plays a physiological role in regulating Cx 5'D-II, we have explored rT3 plasma kinetics, plasma to tissue exchange, and uptake by tissues in the rat, as well as the metabolic routes of degradation and the sources of rT3 in cerebral cortex (Cx). Plasma and tissue levels were assessed with tracer [125I]rT3. Two main compartments were defined by plasma disappearance curves in euthyroid rats (K1 = -6.2 h-1 and K2 = -0.75 h-1). In Cx of euthyroid rats, [125I]rT3 peaked 10 min after iv injection, tissue to plasma ratio being 0.016 +/- 0.004 (SE). In thyroidectomized rats, plasma and tissue [125I]rT3 concentrations were higher than in euthyroid rats, except for the Cx that did not change. PTU caused further increases in all the tissues studied, except for the Cx and the pituitaries of thyroidectomized rats. From the effect of blocking 5'D-I with PTU or reducing its activity by making the animals hypothyroid, we concluded that 5'D-I accounts for most of the rT3 clearance from plasma. In contrast, in Cx and pituitary the levels of rT3 seem largely affected by 5'D-II activity. Since the latter results suggest that plasma rT3 does not play a major role in determining rT3 levels in these tissues, we explored the sources of rT3 in Cx using [125I]T4. The [125I]rT3 (T4) to [125I]T4 ratio remained constant at 0.03 from 1 up to 5 h after injection of [125I]T4. From plasma levels of T4 and rT3, Cx concentration was calculated to be 30 pg rT3/g Cx in euthyroid rats, more than 98% locally produced from T4 deiodination. We conclude that rT3 has a very rapid metabolism, mainly attributed to 5'D-I activity, but that 5'D-II could also play a role in certain tissues. Nearly all rT3 present in Cx is locally derived from T4.

Amniotic fluid thyrotropin (TSH) following maternal administration of thyrotropin releasing hormone.

Cord blood and amniotic fluid thyrotropin (TSH), T4, T3, and rT3 concentrations were measured in 49 women who received 400 micrograms thyrotropin releasing hormone (TRH) iv during labor and in 16 control women who received saline. Cord blood serum TSH concentrations were elevated for as long as 4 hours after TRH administration and peak values (38.0 +/- 4.2 microU/ml) were observed from 61-120 minutes after TSH as compared to control values of 5.0 +/- 0.3 microU/ml. The elevations in fetal TSH concentration stimulated the fetal thyroid, resulting in a progressive increase in cord blood T4 and T3 but not rT3 concentrations. These TRH induced elevations in fetal cord blood TSH concentrations were not accompanied by increases in unconcentrated and 4 fold concentrated amniotic fluid TSH concentrations which were almost always below 0.6 microU/ml, the limit of assay sensitivity. Unconcentrated amniotic fluid T4 concentrations were barely detectable and no variation was observed between the TRH treated and saline treated mothers; amniotic fluid T3 was not detectable in any of the groups; and amniotic fluid rT3 concentrations ranged between 46.4 and 55.6 ng/dl and did not differ between groups. These findings suggest that term amniotic fluid TSH values do not reflect transient but marked elevations in fetal serum TSH concentrations and that amniotic fluid TSH determination is probably not useful in the detection of primary fetal hypothyroidism. It is possible, but unlikely, that long-term and even greater elevations in fetal serum TSH concentrations would result in increased amniotic fluid TSH concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Women on thyroid hormone therapy: pregnancy course, fetal outcome, and amniotic fluid thyroid hormone level.

Thirty-four hypothyroid women on thyroid hormone substitution were followed through 37 pregnancies, and 16 women having previous surgery for thyroid carcinoma and thereafter placed on suppressive thyroxine treatment were followed through 19 pregnancies. The thyroxine treatment needed readjustment in 13 pregnancies (23%) to maintain euthyroidism. At delivery, the maternal free thyroxine index was 126 nmol/L in the group of patients treated for hypothyroidism and 146 nmol/L in the patients with treated thyroid carcinoma. The amniotic fluid thyroxine level in normal pregnancies was 6.7 nmol/L, in hypothyroid patients 6.7 nmol/L, and in patients with thyroid carcinoma 5.6 nmol/L. The amniotic fluid reverse triiodothyronine level in normal pregnancies was 0.51 nmol/L, in hypothyroid patients 0.66 nmol/L, and in patients with thyroid carcinoma 0.70 nmol/L. All infants were euthyroid.

Ontogenesis of placental inner ring thyroxine deiodinase and amniotic fluid 3,3',5'-triiodothyronine concentration in the rat.

Human and rat placentae contain enzymatic activity which converts T4 to rT3 and T3 to 3,3'-diiodothyronine and 3'-monoiodothyronine. This study presents data on the ontogeny of this inner ring iodothyronine deiodinase activity (P-T4ase) in rat placenta. P-T4ase was measured by quantitating the conversion of T4 to rT3 in 700 x g supernatants of placental homogenates. Groups of rats were mated to permit the dams to be killed on the same day, on days 12, 14, 16, 18, and 20 of gestation. Sufficient placental tissue was obtained to measure P-T4ase on all but the 12th day of gestation. The highest level of P-T4ase was observed on day 16. P-T4ase on days 14, 18, and 20 was 52%, 77%, and 41%, respectively, of that observed on day 16 (P less than 0.01, day 16 vs. all other days). Amniotic fluid rT3 concentrations were highest on day 18 and were 61% and 64%, respectively, of that observed on day 18 (P less than 0.01, days 16 and 20 vs, day 18). At 20 days, maternal serum T4 concentrations were significantly lower (P less than 0.01) than on days 14, 16, or 18. A brief period of maternal hypothyroidism (4 or 9 days before the time that the animals were killed on day 20 of gestation) did not significantly alter P-T4ase. These studies indicate that there are age-dependent changes in placental inner ring deiodinase activity in the rat. Amniotic fluid rT3 concentrations may reflect these changes. Brief reductions in maternal serum T4 concentrations do not account for changes in placental inner ring deiodinase activity. These studies emphasize the importance of gestational age in studies of placental inner ring iodothyronine deiodinase.

Direct quantitative estimation of several iodothyronines in rat bile by radioimmunoassay and basal data on their biliary excretion.

A method was developed for the hydrolysis of conjugated iodothyronines in bile with the aid of beta-glucuronidase/arylsulfatase and for subsequent direct estimation of total and free iodothyronines with the aid of specific radioimmunoassay. The amount of conjugated fraction could then be calculated from the difference. Thus, basal biliary excretion of several iodothyronines was measured in 31 normal, fed rats in which the bile duct was drained with polyethylene tubing under pentobarbiturate anesthesia and the bile was collected for 2 h. The free fraction of thyroxine, 3,5,3'-triiodothyronine and 3,3'-diiodothyronine was approx. 30% of total content, while that of 3,3',5'-triiodothyronine and 3,5-diiodothyronine was approx. 20% and that of 3',5'-diiodothyronine was less than 10%. This suggests some considerable differences in the conjugation of individual iodothyronines in the liver. The concentration of T4 in bile was about the same as in plasma, while that of other iodothyronines was about 3-8 times higher than in plasma. This shows close interrelations between the iodothyronine deiodinating pathway in liver cells in vivo and the spectrum of iodothyronine in bile. The average ratio of T3/rT3 as found in bile was about 4.