Urinary metabolites of 14 C-labeled thyroxine in man.

Studies were carried out to determine the chemical structures of thyroxine metabolites after total deiodination. Normal subjects were given thyroxine labeled with (14)C on the nonphenolic ring and the alanine side chain, 8-11 mug/day for 10 days. By paper chromatography of fresh urine, six or more (14)C-labeled compounds were separated. The (14)C-labeled metabolites were concentrated by passing the urine through a nonionic polymeric adsorbent. Two major thyroxine metabolites were identified. The identification was made by three different methods: (a) chromatography, (b) synthesis of derivatives, and (c) recrystallization to constant specific activity. One (14)C-labeled metabolite was identified as thyroacetic acid or 4-phenoxy-(4'-hydroxy) phenyl-acetic acid. Another one was identified as thyronine. Of the total urinary (14)C radioactivity, 43.7% was recovered as thyroacetic acid and 19.8% was recovered as thyronine. Approximately one-fifth of each of these metabolites was present in the urine in bound form which released the free metabolites during acid hydrolysis. The average daily excretion of thyroacetic acid was 13.7% of the renal disposal rate of thyroxine, or approximately 7.5 mug/day. The average daily excretion of thyronine was 6.5% of the renal disposal rate of thyroxine or approximately 3.9 mug/day while the urinary iodide made up 64.7% of the renal disposal rate of thyroxine. Our findings provide the needed proof that the major metabolic pathways of thyroxine remove the iodine atoms by substituting hydrogen for iodine and leave the diphenyl ether nucleus intact.

Fecal and urinary excretion of six iodothyronines in the rat.

Fecal and urinary excretion rates of six iodothyronines were assessed in the rat maintained under normal steady state physiological conditions, to gain a more comprehensive understanding of the mechanisms of control of normal thyroid hormone economy and metabolism. Groups of young adult male rats were injected with trace doses of T4, T3, rT3, 3,3'-diiodothyronine (T2), 3',5'-T2, or 3'-monoiodothyronine, each labeled with 125I, and feces and urine were collected separately for up to 10 days. Pooled fecal pellets were homogenized in saline, extracted in ethanol, evaporated under vacuum, and reconstituted in NaOH. Fecal extracts and urine were chromatographed on Sephadex G25 columns under conditions providing quantitative separations of components of interest. A new technique was also developed, based on a model of the in vitro extraction and measurement process, to correct chromatographic results for possible variable recoveries and possible artifactious degradation of radioactively labeled components. No iodothyronines or their conjugates were excreted in urine; all radioactivity was in the form of iodide. In feces, about 30% of the [125I]T3 injected was excreted as T3; and 24% of the [125I]T4 injected was excreted as T4, plus 4% as T3. Together, these results imply that about 24% of endogenous T4 production is excreted as T4 and 76% is irreversibly metabolized; and for T3, about 30% of endogenous T3 production is excreted as T3 and 70% is degraded. For the nonhormonal iodothyronines, about 6% of injected monoiodothyronine, 3% of injected 3',5'-T2, 2% of injected 3,3'-T2, and less than 1% of injected rT3 were excreted in feces as such, indicating that these substances are nearly completely deiodinated in vivo. Very little (1-7%) iodide was excreted as such in feces, which also were devoid of measurable conjugates. An open question is whether the substantial wastage of thyroid hormones in feces represents poor hormone economy in the usually accepted sense or a functional property of overall thyroid hormone regulation.

The role of thyronine in thyroid hormone metabolism.

Thryonine (T0) has been identified in human urine using gas chromatography-mass fragmentography (G.C.M.F.). In 22 normal individuals urinary T0 concentration was found to range between 8--25 nmol/24h. Assuming the mean normal thyroxine (T4) production rate to be approximately 100 nmol/24h, our findings indicate that less than 20% of this could be accounted for as urinary T0 excretion, thus supporting earlier findings that the peripheral metabolism of T4 is not limited solely to deiodination.

Urinary excretion of free and conjugated 3',5'-diiodothyronine and 3,3'-diiodothyronine.

RIAs for the estimation of 3',5'-diiodothyronine (3',5'-T2) and 3,3'-diiodothyronine (3,3'-T2) in human urine have been established. The urinary excretion of both glucuronide and sulfate conjugates of T2 and of T4, T3, and rT3 were estimated by means of enzymatic deconjugation. In healthy controls, the mean excretion (picomoles per 24 h) of free T4 was 1820, that of free T3 was 813, that of free rT3 was 77, that of free 3',5'-T2 was 13, and that of free 3,3'-T2 was 674. The total excretion of free and conjugated T4 was 2941, that of T3 was 1283, that of rT3 was 791, that of 3',5'-T2 was 709, and that of 3,3'-T2 was 2688. Significant amounts of sulfated T4 and T3 could not be demonstrated, amounts of sulfated T4 and T3 could not be demonstrated, whereas the excretion of sulfated rT3 was higher than that of glucuronidated rT3 (P less than 0.001). In contrast, glucuronidated and sulfated 3',5'-T2 as well as glucuronidated and sulfated 3,3'-T2 were found in the urine in equal amounts. In hyperthyroidism, the excretions of free and glucuronidated iodothyronines were increased, whereas the increase of the excretions of sulfated iodothyronines were less pronounced, only reaching statistical significance for 3,3'-T2 (P less than 0.02). In hypothyroidism, the excretions of both free, glucuronidated and sulfated iodothyronines were reduced. Significant amounts of sulfated T4 and T3 could not be demonstrated in urine from hyperthyroid or hypothyroid patients. Our data demonstrate that the amounts of free iodothyronines excreted in the urine vary considerably, suggesting active renal handling. The amounts of urinary glucuronidated and sulfated conjugates of the different iodothyronines studied vary considerably and are affected by thyroid function.

3,5,3'-Triiodothyronine (T3) sulfate: a major metabolite in T3 metabolism in man.

Previous studies have suggested that T3 metabolism relies more on nondeiodinative conjugation than on direct deiodinative degradation for its disposal in man. To better define this process, tracer T3 kinetic studies were performed in five euthyroid subjects before and after iopanoic acid (IA) administration to selectively impair T3 deiodinative disposal. Both a low IA (0.5-g load, followed by 0.5 g/day for 7 days) and a high IA (3.0-g load, followed by 3.0 g/day for 7 days) dosing schedule were employed to achieve varying levels of deiodinase inhibition. Additionally, the high IA dose was repeated with simultaneous oral T3 administration (100 micrograms daily) to normalize serum T3 levels that were reduced by IA-induced inhibition of T4 to T3 conversion. The results demonstrated that baseline serum T3 (2.3 +/- 0.1 nmol/L) and T3/T4 (1.9 +/- 0.1 x 10(-2)) values were significantly reduced by both the low IA (1.5 +/- 0.1 nmol/L and 1.2 +/- 0.1 x 10(-2), respectively) and the high IA (1.5 +/- 0.1 nmol/L and 0.9 +/- 0.2 x 10(-2), respectively) dosing schedule and that the addition of oral T3 to the high IA regimen restored both the T3 and T3/T4 levels to near-normal values (2.9 +/- 0.3 nmol/L and 1.7 +/- 0.2 x 10(-2), respectively). Low IA also significantly decreased T3 clearance (30 +/- 4 to 18 +/- 2 L/day; P < 0.005) and fractional urinary tracer recovery (70 +/- 3% to 37 +/- 4%; P < 0.005), whereas high IA produced only a minimal further reduction in clearance (16 +/- 2 L/day; P < 0.01) and urinary tracer recovery (32 +/- 3%; P < 0.05). Surprisingly, oral administration of T3 to the high IA regimen significantly increased T3 clearance (23 +/- 4 L/day; P < 0.01) without changing urinary tracer recovery (34 +/- 5%) compared to the effects of high IA alone. Evaluation of the urinary T3 metabolite pattern demonstrated that the major products of T3 metabolism were T3 sulfate and 3,3-diiodothyronine sulfate. These observations confirm previous results suggesting that the majority of nondeiodinative T3 disposal occurs via T3 sulfate formation. The additional finding that such nondeiodinative disposal may also be influenced by the circulating T3 level leads us to propose that sulfotransferase enzyme systems may play an important role in regulating the prereceptor availability of this ligand.

A radioimmunoassay for measurement of thyronine and its acetic acid analog in urine.

A sensitive and specific RIA has been developed to measure thyronine (To) in urine. The RIA used an anti-To antibody obtained from a rabbit immunized with a L-To-human serum albumin conjugate and [3H]To as the radioligand. The acetic acid analog of To (ToAc), that is the diphenyl structure with an acetic acid side-chain, cross-reacted strongly with the antibody. Relative to To, it cross-reacted 160% in phosphate-buffered saline, pH 7.4, and 100% in 0.075 mol/L barbital buffer, pH 8.6, containing sodium salicylate (final concentration, 8 mg/mL). The latter conditions were employed for the RIA, and the results reported thus reflect the presence of To and/or ToAc. 3-Monoiodothyronine, 3'-monoiodothyronine, 3',5'-diiodothyronine, and 3,5-diiodothyronine cross-reacted with the anti-To antibody 1.9%, 1.7%, 0.3%, and 0.2%, respectively; the cross-reactivity of other To derivatives and tyrosine and its derivatives was less than 0.05%. Urinary To and/or ToAc excretion in 12 normal subjects averaged 16 +/- 2 (+/- SE) micrograms/day (59 +/- 9 nmol/day) or 14 +/- 2 micrograms/g creatinine (5.9 +/- 0.6 nmol/mmol creatinine). Treatment of urine from normal subjects with beta-glucuronidase or sulfatase did not significantly alter the To content. Column and thin layer chromatographic studies revealed that 83% and 61%, respectively (range, 37-100%), of urinary To immunoreactivity was attributable to ToAc. The mean daily excretion of To in 20 patients with nonthyroidal illness [NTI; 22 +/- 4 micrograms/day (82 +/- 17 nmol/day)] was similar to that in normal subjects, but was elevated when expressed as nanomoles per mmol creatinine (20 +/- 2; P less than 0.001), because creatinine excretion was reduced in the NTI patients. The mean daily urinary To excretion in 13 patients with hyperthyroidism due to Graves' disease was slightly elevated [29 +/- 6 micrograms/day (108 +/- 21 nmol/day); P less than 0.1], but was clearly elevated when expressed as nanomoles per mmol creatinine (37 +/- 8; P less than 0.001), again because creatinine excretion was reduced in these patients. The mean urinary To excretion was subnormal in 13 patients with hypothyroidism and was significantly (P less than 0.005) less than that in the NTI patients regardless of the manner in which the results were expressed. Analysis of pronase hydrolysates of thyroid glands obtained at autopsy from euthyroid patients suggested that the To content of the thyroid approximates only 1.2% that of T4, supporting the thesis that prior iodination of tyrosine is critical for the coupling process in the thyroid.(ABSTRACT TRUNCATED AT 400 WORDS)

Radioimmunoassay for 3,3'-diiodothyronine in human serum.

A specific radioimmunoassay for measurement of 3,3'-diiodothyronine (T2') is presented. With the method described (ethanol extraction of native serum and lyophilisation of the extract) the application of 400 microliter serum equivalent in the assay is possible. Standards and sera are treated similarly. The detection limit is 0.625 ng/dl, comparison between direct assay and dried extract assay shows good correlation. Mean normal T2' serum concentration in man is 7.2 ng/dl (range 3 to 11 ng/dl), hypothyroid: below 3.0 ng/dl, hyperthyroid: 11-64 ng/dl (range). T2' level in cord-blood of newborns: 16.5 ng/dl. The urinary excretion of free T2' of normal man is 0.49 microgram/24 h (mean), a relatively high excretion rate in comparison to the low serum level.

Mass spectral properties of volatile derivatives of thyronine (T0) and use of these in the study of thyronine excretion in eu-, hyper- and hypothyroidism.

The mass spectral properties of four classes of derivatives of thyronine are discussed ( oxazolidinone , O-methyl oxazolidinone , O-acetyl oxazolidinone and N,O- diheptafluorobutyryl methyl ester). An assay for thyronine in human urine is described based on the N,O- diheptafluorobutyryl methyl ester. Results of T0 excretion in euthyroid humans were compared with those obtained previously using an assay based on the O-acetyl oxazolidinone derivative. Patients with frank hyperthyroidism had significantly higher T0 excretion than euthyroid subjects (2 alpha less than 0.002) and hypothyroid patients lower T0 excretions than euthyroid subjects (2 alpha less than 0.002). Some overlap between the two pathological ranges and the normal euthyroid range was evident.

3,5-Diiodotyrosine and thyronine in the urine of patients with chronic renal disease.

Urinary 3,5-diiodotyrosine (DIT) and thyronine (T0) excretion was investigated in 18 patients with chronic renal disease. In accord with previous findings serum T4 and thyroid hormone binding proteins measured in 17 patients were in the low or normal range. Urinary albumin excretion was elevated in all 18 and T4 binding prealbumin (TBPA) in 15 of the 18. Urinary T0 excretion measured in 12 patients was also significantly lower than normal (mean +/- SD 4.4 +/- 2.6 vs 15.8 +/- 5.8 nmol/24 h renal vs normal 2 P less than 0.001). In contrast urinary DIT excretion was significantly elevated in renal patients compared with normal subjects (2.0 +/- 1.5 vs 0.75 +/- 0.41 nmol/24 h, respectively). Possible sources of the increased DIT are discussed.

Urinary excretion of unconjugated and conjugated 3,5-diiodothyronine.

A radioimmunoassay for the estimation of 3,5-diiodothyronine (3,5-T2) in human urine has been established. The urinary excretion of both glucuronide and sulfate conjugates of 3,5-T2 were estimated after enzymatic deconjugation. In 19 healthy controls the median excretion of unconjugated 3,5-T2 was 276 pmol/d, whereas the median excretion of glucuronidated and sulfated 3,5-T2 in 7 healthy subjects was 448 and 451 pmol/d, respectively. The median excretion of 154 pmol/d in 9 hypothyroid patients did not differ from that found in controls. In contrast 12 patients with hyperthyroidism had an enhanced excretion, 1312 pmol/d (P less than 0.01). Compared with previous data on the daily degradation of 3,5-T2, it is concluded that approximately one-sixth of degradated 3,5-T2 is excreted in the urine.

Renal handling of iodothyronines in acromegaly.

Measurement of the free serum concentration, the 24-h urinary excretion and the renal clearance of T4, T3, 3,3',5'-tri-iodothyronine (rT3), 3',5'-diiodothyronine (3',5'-T2) and 3,3'-di-iodothyronine (3,3'-T2) was performed in 13 patients with active acromegaly and in 18 healthy controls. The acromegalic patients had normal serum levels of the free iodothyronines, whereas the urinary excretion of T4 and T3 was increased approximately two-fold in the patients with acromegaly. The creatinine clearance, reflecting the glomerular filtration rate (GFR), was increased in the acromegalic patients, in median 133 ml/min versus 87 ml/min (p less than 0.01) in the controls. Compared to the creatinine clearance the clearance of T3 and 3,3'-T2 was higher (p less than 0.01) in acromegalics as well as in controls. The patients with acromegaly had higher renal clearance of T4 and T3 than controls, in median 81 ml/min versus 33 ml/min, and 269 ml/min versus 137 ml/min, respectively (p less than 0.01). These differences were not due to changes in creatinine clearance. The renal clearance of 3',5'-T2 tended to be enhanced in acromegalic patients (8.2 ml/min versus 3.9 ml/min, p less than 0.10), both before and after correction for creatinine clearance. The data suggest that in acromegaly, as in normal condition, iodothyronines are subject to both glomerular filtration and active tubular transport mechanisms. Further, active acromegaly results not only in increased GFR, but also in changes of the net tubular transport in favour of secretion of at least T4 and T3, and possibly also of 3',5'-T2.