3,3'-Diiodothyronine sulfate excretion in maternal urine reflects fetal thyroid function in sheep.

We have shown that there is significant fetal-to-maternal transfer of sulfated metabolites of thyroid hormone after fetal infusion of a pharmacologic amount of 3,3',5-triiodothyronine (T(3)) or sulfated T(3) in late pregnancy in sheep (Am J Physiol 277:E915, 1999). The transferred iodothyronine sulfoconjugate, i.e. 3,3'-diiodothyronine sulfate (T(2)S), of fetal origin appears in maternal sheep urine. The present study was carried out to assess the contribution of T(2)S of fetal origin to the urinary pool in ewes. Eighteen date-bred ewes (mean gestational age of 115 d) and their twin fetuses were divided into four groups. In group I (control, n = 5), both ewes (M) and their fetuses (F) were sham operated for thyroidectomy (Tx). In group II, the ewes (MTx, n = 4) and, in group III, the fetuses (FTx, n = 4) were subjected to Tx. In group IV (MTx.FTx, n = 5), both the ewe and fetus had Tx. After 10-12 d, fetal and/or maternal hypothyroidism were confirmed by serum thyroxine (<15 nmol/L) measurements. In addition, we infused radioactive T(3) without disturbing the T(3) pool in three singleton near-term fetuses and assessed the amount of radioactive iodothyronine that appeared in maternal urine (MU). After infusing [(125)I-3'],3,5-T(3) via fetal vein to the near-term normal fetuses, radioactive T(2)S was identified as the major metabolite in MU by HPLC and T(2)S-specific antibody. MU T(2)S excretion (pmol/mmol creatinine) was significantly reduced by FTx and MTx.FTx but not by MTx. In addition, positive correlations (p < 0.01) were found between MU T(2)S excretion and fetal serum thyroxine and T(3) concentrations but not with maternal serum thyroxine or T(3) levels. T(2)S of fetal origin contributes significantly to the MU pool.

Iodothyronine sulfotransferase activity in rat uterus during gestation.

In developing mammals, we and others demonstrated that sulfation is an important pathway in the metabolism of thyroid hormone, and there is significant fetal-maternal transfer of sulfated iodothyronine. In the present study, we characterized a novel iodothyronine sulfotransferase (IST) in pregnant rat uterus. (125)I-labeled 3,3'-diiodothyronine (T(2)), T(3), rT(3), and T(4) were used as substrates with unlabeled 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the sulfate donor. Sulfated iodothyronine products were separated by Sephadex LH-20 column and further identified on reverse phase HPLC. We measured IST activity in pregnant rat uterus by incubating 1 microM substrate, 50 microM PAPS, and 50 microg cytosol protein, pH 7.2, 30 min at 37 degrees C. The results show that the substrate preference of the uterine IST activity is: T(2 )> rT(3 )> T(3)> T(4); the pH optimum is 6.0 for T(2). The K(m) and V:(max) (for gestational day 21 uterus) for T(2) are 0.62 microM and 3466 pmol/mg protein/h, respectively; for PAPS the values are 2.6 microM and 1523 pmol/mg protein/h, respectively. During pregnancy, the total activities exhibit a U-shaped curve with minimum activity at day 13 of gestation; while a thermostable activity increases significantly near term. In summary, there is significant uterine IST that varies during pregnancy. The role of this uterine sulfotransferase activities in regulating the bioavailability of thyroid hormone in the developing fetus remains to be elucidated.

Enterohepatic regulation and metabolism of 3,5,3'-triiodothyronine in hypothyroid rats.

Several steady state indices of thyroid hormone distribution, metabolism, excretion, and absorption were measured in intact hypothyroid and euthyroid rats, to explore the role of intestines and enterohepatic pathways in the dynamic regulation of whole-body thyroid hormone in these two states. Ten rats were studied, 5 normal control (N) and 5 rendered hypothyroid (3.48 vs. 19.8 ng/ml TSH) by surgical thyroidectomy 3.5 weeks earlier (HYPO). High specific activity 125I-labeled T3 (T3*) was infused at the same constant rate for 7 days from osmotic minipumps implanted sc. Daily urine and feces, and seventh-day cardiac and portal venous blood, bile, and whole intestinal contents were assessed. Bowel and feces were homogenized, extracted, and chromatographed, along with serum, bile, and urine samples. Bile, bowel, and fecal extract samples were also hydrolyzed with aryl-sulfatase and/or beta-glucuronidase and chromatographed to identify conjugates and determine total T3* in all fluid and tissue samples. In the N group, the bowel contained 21.2 +/- 1.22 (SD) times more T3* (mass) than plasma (199 ng vs. 9.39 ng), this ratio falling to 9.03 +/- 1.78 in the HYPO group (30.4 ng vs. 3.37 ng), a shift to relatively more T3* in blood. Urinary T3* was zero in both groups. But fecal excretion was 34 +/- 4.43% of total T3* infused (production) in N and only 20.3 +/- 3.05% in HYPO rats, closely paralleling reduced fecal bulk flow, and thus providing more time for T3* absorption. Endogenous T3 and T4 concentrations measured in portal plasma were 15-31% greater in normals and 69-95% greater in HYPO rats than in corresponding systemic plasma samples, a direct indication of absorption of endogenous T3 and T4 in both groups, with greater absorption in the HYPO group. About 66% total T3* was metabolically degraded in N rats, rising to approximately 80% in HYPO rats. Plasma clearance rates of T3 fell more than 50% in HYPO rats, and total T3 production fell to about 20% of normal. It appears that HYPO rats compensate for low T3 by fecally excreting a much smaller fraction of total T3 production, absorbing more T3 and T4, and leaving a larger fraction for T3 action and degradative metabolism.

Identification of thyroxine-sulfate (T4S) in human serum and amniotic fluid by a novel T4S radioimmunoassay.

Recently, we identified significant amounts of thyroxine sulfate (T4S) in fetal sheep serum, meconium, bile, and amniotic and allantoic fluids. Little is known, however, about sulfate conjugation of thyroxine in humans. In this study, we employed a novel, sensitive T4S RIA to address this question. The rabbit antiserum was quite specific; T4, T3, rT3, and 3,3'-T2 showed less than 0.002% cross-reactivity. Other analogs cross-reacted less than 0.0001%. Only rT3S and T3S cross-reacted significantly (9.9% and 2.0%, respectively). The mean serum T4S concentration (ng/dL) was 8.6 in euthyroid subjects, 14.4 in hyperthyroid subjects, 5.0 in hypothyroid subjects, 5.9 in pregnancy, and 4.5 in patients with nonthyroid illnesses. T4S concentration in amniotic fluid from women at 18-19 weeks of gestation (25.5 ng/dL) was higher than that at 14-15 weeks of gestation (14.3 ng/dL). A significant rise in serum T4S was detected in hyperthyroid patients 1 day after ingestion of 1 g of ipodate. These data suggest that T4S is a normal component of human serum and amniotic fluid, and it is mostly derived from T4 peripherally and accumulates when type I 5'-monodeiodinating activity is low in fetuses or inhibited by drugs, such as ipodate.

Further studies on the long-term treatment of Graves' hyperthyroidism with ipodate: assessment of a minimal effective dose.

We have previously described that sodium ipodate (500 mg/day, p.o.) is effective in normalizing serum T3 and T4 levels in most patients with Graves' hyperthyroidism. In this study, we examined serum T3, T4, and rT3 levels in 14 hyperthyroid patients with Graves' disease during treatment with a lower dose (500 mg, every other day, p.o.) of sodium ipodate for a period of 3-30 weeks (mean 15.5 weeks). Three types of responses were observed. In group I (4 patients), both serum T3 and T4 were in the normal range at the end of treatment [baseline: mean +/- SEM T3, 6.8 +/- 0.96 nmol/L (normal 0.92-3.0)] and T4 [256 +/- 44 nmol/L (normal 62-167); post-ipodate: T3, 2.0 +/- 0.46 nmol/L and T4 107 +/- 28 nmol/L]. In group II (n = 5), either serum T3 (3 patients) or serum T4 (2 patients) did not become normal (baseline: T3 7.7 +/- 1.1 and T4 228 +/- 3.9; post-ipodate: T3 2.9 +/- 0.57 and T4 188 +/- 27 nmol/L). In group III (5 patients), neither serum T3 nor serum T4 returned to normal following ipodate treatment (baseline: T3 11.9 +/- 1.8 and T4 260 +/- 23; post-ipodate: T3 7.5 +/- 0.49 and T4 322 +/- 17 nmol/L). The mean serum rT3 concentration increased during ipodate treatment to a peak value of 100% above baseline and remained elevated (20-75% above baseline) throughout the study. Some improvement in hyperthyroidism was suggested by increase in body weight during ipodate treatment in most cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of type II T4-5'-monodeiodinase activity in brown adipose tissue in fasted mice.

In order to study the effect of starvation on brown adipose tissue (BAT) type II 5'-monodeiodinating activity (5'MDI), type II 5'MDI was measured in vitro in the presence of 20 mM dithiothreitol, 1 mM propylthiouracil, 2 nM thyroxine (T4) and appropriate amounts of 600 X g infranatant of BAT from fed control or 3 day fasted mice, with or without daily T4 replacement (1.2 micrograms/100 g bw) during starvation. I- released from 125I-T4 was measured by ion-exchange column chromatography. Activity of BAT 5'MDI was markedly elevated in the 3 day fasted group (133 +/- 28 fmol I-/h per mg protein vs. 26 +/- 6.4; p less than 0.05). Kinetic studies using BAT infranatant suggested that fasting-induced activity is associated with a similar change in the Vmax, but no demonstrable change in apparent Km of T4 monodeiodination. T4 replacement during fasting, which normalized both serum T4 and T3 in fed and 3 day fasted groups, did not stop the increase of BAT 5'MDI in the fasted group (p less than 0.01). The data suggest that: (1) the fasting-induced increase in BAT 5'MDI is due mainly to the changes in capacity rather than the affinity of the enzyme, and (2) the fasting-induced increase in BAT 5'MDI is not mediated entirely through changes in serum thyroid hormone concentration.

Stimulation of thyroidal iodothyronine 5'-monodeiodinase by long-acting thyroid stimulator (LATS).

To evaluate the effect of long-acting thyroid stimulator (LATS) on thyroid iodothyronine monodeiodinating activity, we have studied the in vitro conversion of T4 to T3 by mouse thyroid homogenate comparing tissue from LATS treated (0.1 ml LATS(+) serum, ip, for 3 days) with tissues from LATS(-) Graves' disease patients' serum or normal serum treated controls. Five out of seven LATS(+) sera were shown to stimulate the T4 5'-deiodinase significantly in mouse thyroid. There was no significant correlation between LATS titre and deiodinase activities in the different sera tested. To compare the effect of LATS and TSH (0.2 IU, ip daily), studies were carried out from 12 to 72 h. LATS had a similar latency of 12 h on the stimulation of thyroid deiodinase compared to TSH as reported earlier. However, the conversion activities reached a plateau by 12 h after LATS treatment, while it continued to rise upon daily TSH injection from 24 to 72 h. In addition, TSH caused a marked reduction of thyroid protein and an early peaking in serum T3 and T4 at 12 h, whereas LATS caused no detectable change in thyroid protein and a gradual rise in circulating T3 and T4. The kinetic analysis indicated that LATS-mediated stimulation of T4 5'-deiodinase was, similar to TSH, associated with an increase in maximum velocity (Vmax were 139, 208 and 505 pmol/mg protein/30 min respectively in control, LATS and TSH-treated animals) without a demonstrable change in the apparent Km (approximately 2.0 microM for T4). The present study demonstrated that some LATS-rich sera stimulate thyroid T4 to T3 conversion in mouse. It provides an insight into the mechanism of increased T3 secretion from Graves' thyroid glands.

Characterization of thyrotropin-induced increase in iodothyronine monodeiodinating activity in mice.

To further characterize the effect of TSH administration on thyroid iodothyronine monodeiodinating activity, we have evaluated the in vitro conversion of T4 to T3 (outer ring deiodination) and T3 to 3,3'-diiodothyronine (T2; inner ring deiodination) by mouse thyroid, liver, and kidney homogenates, comparing tissues from TSH-treated mice (0.1-200 mU bovine TSH, ip, for 1-3 days) with tissues from saline-treated controls. The in vitro conversion activity was studied in the presence of 1-20 mM dithiothreitol; most of the studies were carried out at 4 mM. Studies were carried out at optimal pH 6.5 for outer ring and 7.8 for inner ring deiodination. The iodothyronine monodeiodinase in mouse thyroid is similar to the ones in liver and kidney. It is heat labile (inactivated at 56 C for 5 min), inhibited by propylthiouracil (0.2 mM) and ipodate (0.2 mM), and unaffected by methimazole (up to 20 mM), ascorbate (up to 0.1 M) or KI (up to 20 mM). The mean +/- SE baseline rates of T4 to T3 and T3 to T2 conversion were 100 +/- 6.3 and 56.5 +/- 2.9 pmol/mg thyroid protein X 30 min at 37 C, respectively. A significant increase in each conversion activity was found after TSH treatment (0.2 U, ip, daily for 3 days); T4 to T3 conversion rose to 282 +/- 15.4, and T3 to T2 increased to 153 +/- 7.4 pmol/mg thyroid protein (P less than 0.001). A 12.8% increase in thyroid weight was found in the TSH-treated group (P less than 0.03 compared with saline control group). Similar but less marked increased in monodeiodinating activities were seen in the liver. A minimal but significant increase in inner ring monodeiodination with no significant increase in T4 to T3 converting activity was found in kidney, which, in the mouse, has markedly less outer ring deiodinase than liver or thyroid. The iodothyronine monodeiodinating activities did not increase until 12 h in thyroid and 48 h in liver after the first dose of TSH. Significant increases in T4 to T3 and T3 to T2 conversion were seen with doses of TSH as low as 0.1 mU (ip, daily for 3 days), and there was a linear dose-response thereafter. The decay of the increased iodothyronine monodeiodinating activities after a single dose of TSH (0.2 U) appeared to be linear, with a decay t 1/2 of 1.3 days for T4 to T3 conversion and about 1.0 day for T3 to T2 conversion.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute effects of iodine on the stimulated rat thyroid.

Rats fed a low iodine diet (LID) for 5 weeks were given 125I as NaI to prelabel their thyroids; 24 h later, they were injected ip with 1 mg KI. They were then killed at different time intervals of up to 4 h. Thyroids of control animals not injected with KI were hyperplastic, the tissue showed microfollicles devoid of colloid, thyroglobulin was poorly iodinated, and thyroid peroxidase activity was increased. Within 4 h after KI injections, there was 1) a decrease in thyroid weight, 2) expansion of the follicular lumens of the hyperplastic in thyroid tissue and accumulation of colloid, 3) an increase in the thyroglobulin iodine concentration, and 4) a decrease in the thyroid peroxidase activity. Some of these changes started to occur as early as 60 min after KI administration. By that time, using the microprobe, most of the organically bound iodine was detectable inside the follicular cells. Radioiodine (125I) used to prelabel the thyroids was accumulated inside the follicles during the entire observation period. The serum TSH level was elevated and did not decrease after KI administration. It is concluded that excess iodine in LID rats may produce acute morphological and biochemical changes which seem to be a direct effect of iodine not mediated by modifications of TSH. The distribution of organically bound iodine, as determined by the microprobe, and the biochemical studies were interpreted as suggesting intracellular thyroglobulin iodination in these stimulated rat thyroids.