Sephadex LH-20 column separation of thyroidal iodoamino acids

Williams, A.; Freeman, Donna E.; Florsheim, Warner H.

doi:10.1016/s0021-9673(01)86233-6

Cited by 40 publications

(13 citation statements)

References 20 publications

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“…J.) (15). Purity of the labeled T3 eluted was verified in a tate; T3, triiodothyronine; T4, thyroxine; TBG, thyroxinebinding globulin; TBPA, thyroxine-binding prealbumin; TETRAC, tetraiodothyroacetic acid; TLC, thin-layer chromatography.…”

Section: Methodsmentioning

confidence: 99%

Thyroid Hormone Binding by Human Serum Prealbumin (TBPA). ELECTROPHORETIC STUDIES OF TRIIODOTHYRONINE-TBPA INTERACTION

Davis¹,

Handwerger²,

Gregerman³

1972

J. Clin. Invest.

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Section: Methodsmentioning

confidence: 99%

Thyroid Hormone Binding by Human Serum Prealbumin (TBPA). ELECTROPHORETIC STUDIES OF TRIIODOTHYRONINE-TBPA INTERACTION

Davis¹,

Handwerger²,

Gregerman³

1972

J. Clin. Invest.

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“…A tracer quantity of T4-AI (Abbott Laboratories) was added at the outset (10,000 dpm/ng of T4). The resultant mixture was purified by column chromatography on Sephadex LH 20 (30) followed by five successive paper chromatographic separations in a t-amyl alcohol system (18) until a constant specific activity had been achieved. Gas chromatographic analysis (10) of the purified T4 preparation indicated that its T3 content was less than 0.01%.…”

Section: Detailed Proceduresmentioning

confidence: 99%

Serum triiodothyronine: measurements in human serum by radioimmunoassay with corroboration by gas-liquid chromatography

Mitsuma¹,

Nihei²,

Gershengorn³

et al. 1971

J. Clin. Invest.

102

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A B S T R A C T Serum triiodothyronine (T3) has been measured by radioimmunoassay and corroborated by analysis of the identical samples with a previously described gas-liquid chromatographic technique. Special features of the radioimmunoassay procedure which permit determinations in unextracted serum include the use of a T3-free serum preparation for the construction of the standard curve and of tetrachlorothyronine to inhibit binding of T3 to thyroxine-binding globulin. T3 values by radioimmunoassay were 138 ±23 ng/100 ml (mean +SD) in 82 normal subjects, 62 ±9 ng/100 ml in 45 hypothyroid patients, and 494 ±265 ng/100 ml in 60 patients with toxic diffuse goiter. In the hypothyroid group, the range was similar in patients with both primary and secondary hypothyroidism. There was no overlap between the three thyroidal states. Elevated T3 levels were seen in 40 cases that appeared clinically hyperthyroid but had normal serum thyroxine (T4) de-terminations, a syndrome we have called T3 toxicosis. Values obtained with radioimmunoassay agreed closely with those we had previously found by gas-liquid chromatography which were 68 ±2 ng/100 ml in hypothyroidism, 137 ±23 ng/100 ml in normal subjects, and 510 ±131 ng/100 ml in untreated toxic diffuse goiter.Since T3 is very potent and its level varies in different clinical states, accurate T3 measurements are required to assess a patient's thyroid status properly. The radioimmunoassay for T, appears to be sufficiently sensitive, precise, and simple to permit its routine clinical application for this purpose.

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“…We adapted a separation method reported by Williams and co-workers (24). Sephadex LH-20 was equilibrated in ethyl acetate-methanol-2 n NH 4 OH (400:100:40, vol/vol/vol) eluent for 24 h, and the fines were discarded by decantation.…”

Section: T 3 ** Sephadex Lh-20 Purificationmentioning

confidence: 99%

Direct Measurement of the Contributions of Type I and Type II 5'-Deiodinases to Whole Body Steady State 3,5,3'-Triiodothyronine Production from Thyroxine in the Rat

Nguyen

1998

Endocrinology

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Production of T 3 from T 4 in tissues is catalyzed by two 5Ј-deiodinases, type I (D1) and type II (D2), but the quantitative contribution of each pathway to whole body T 3 production is not well established. In the presence of propylthiouracil (PTU), D1, but not D2, can be effectively blocked, providing an experimental probe for addressing this problem. Decades ago, this approach provided indirect estimates ranging from 23-44% contribution by D2, based on plasma T 3 appearance rate comparisons (PAR 3 ϭ PCR 3 [T 3 ] p ) in periodically T 4 -injected athyreotic rats vs. controls. Two, more recent studies, using constant infusions of T 4 for replacement, achieved 22% and 65% estimates, respectively, from PAR 3 comparisons. We have revisited this problem more directly and precisely, with two major differences in experiment design. We used direct whole body steady state measurements of T 3 production, instead of indirect plasma-only data (PAR 3 ). We also used (euthyroid) physiological doses of both T 4 (0.9 g/day⅐100 g BW) and T 3 (0.15 g/day⅐100 g BW) for replacement in two thyroidectomized rat groups, instead of T 4 only, in a 7-day constant steady state, dual tracer infusion protocol. The first group also had chronically implanted 150-mg PTU pellets (TXR-PTU); the other had implanted 0.1 N NaOH placebo pellets (TXR-EU); each delivered their product at constant rates. A third euthyroid intact group was used as the controls. The completeness of D1 inhibition was ascertained in a fourth group, identically treated with 150-mg PTU pellets, in which negligible D1 activity was found in liver and kidney using labeled rT 3 as substrate for the 5Ј-D assays and minimal (1 mM) dithiothreitol as cofactor. In the TXR-PTU group, the percentage of T 4 converted to T 3 was 11.8%, compared with 23.4% (P Ͻ 0.0005) in the TXR-EU group, and 22.7% (P ϭ NS) in controls. Thus, in euthyroid steady state, D2 contributes about half of the T 3 produced from T 4 . (Endocrinology 139: 4626 -4633, 1998) I T IS NOW well established that T 3 production from T 4 is locally regulated and catalyzed by at least two tissuespecific 5Ј-deiodinase enzymes, type I (D1) and type II (D2), distinguishable by several factors. D1 is highly sensitive to inhibition by 6-propyl-2-thiouracil (PTU) and other thiols (1), with a K m in the nanomolar range with physiological cofactors such as glutathione (2), and its affinity is higher for rT 3 than for T 4 (1). D1 is expressed in many tissues, but the highest levels are found in liver, kidney, and thyroid in humans and rodents (3). In contrast, D2 is relatively insensitive to PTU, it also has a K m in the nanomolar range, and its affinity is higher for T 4 than for rT 3 (1). In rodents, D2 is expressed primarily in the central nervous system, anterior pituitary, and brown adipose tissue (4 -6) and in humans in the central nervous system (7), placenta, skeletal muscle, heart, and thyroid (8 -10).D1 and D2 activities are strongly correlated with thyroid hormone status. D2 activity is increased in hypothyro...

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Sephadex LH-20 column separation of thyroidal iodoamino acids

Cited by 40 publications

References 20 publications

Thyroid Hormone Binding by Human Serum Prealbumin (TBPA). ELECTROPHORETIC STUDIES OF TRIIODOTHYRONINE-TBPA INTERACTION

Thyroid Hormone Binding by Human Serum Prealbumin (TBPA). ELECTROPHORETIC STUDIES OF TRIIODOTHYRONINE-TBPA INTERACTION

Serum triiodothyronine: measurements in human serum by radioimmunoassay with corroboration by gas-liquid chromatography

Direct Measurement of the Contributions of Type I and Type II 5'-Deiodinases to Whole Body Steady State 3,5,3'-Triiodothyronine Production from Thyroxine in the Rat

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