Total and free triiodothyronine in human serum

A reliable method has been developed for the determination of total serum T3, dialyzable fraction (DFT3), and absolute concentration of free T3 (AFT3). Total T3 values (mean ± SD) were: healthy euthyroid subjects, 0.33 ± 0.07 μg per 100 ml; hyperthyroid patients, 0.71 ± 0.1 μg per 100 ml; hypothyroid, 0.10 ± 0.03 μg per 100 ml. Values (mean ± SD) for DFT3 in these groups were 0.46 ± 0.14%, 0.78 ± 0.17%, and 0.16 ± 0.08%, respectively. Calculated values for AFT3 were: 1.51 ± 0.4 mμg per 100 ml, 5.00 ± 0.6 mμg per 100 ml and 0.24 ± 0.1 mμg per 100 ml, respectively. Dilution of serum before dialysis lowered estimated DFT3 values. Enrichment of serum with labeled T3 in the range examined did not affect DFT3. However, DFT3 was increased by addition of Merthiolate to serum in concentration 1: 10,000 due to displacement of T3 from thyroxine-binding globulin to albumin. The data suggest that triiodothyronine may play a considerably more important role in normal and pathological physiology, as evidenced by kinetic analysis using these data. A metabolic role for T3 equal to that of T4 is indicated.     

Salicylate-induced increases in free triiodothyronine in human serum: Evidence of inhibition of triiodothyronine binding to thyroxine-binding globulin and thyroxine-binding prealbumin

Addition of sodium salicylate to human serum at concentrations often obtained during aspirin therapy causes 100-200% increases in free triiodothyronine (T(3)) and free thyroxine (T(4)) as estimated by ultrafiltration. The increase in free T(3) was unexpected since previous data had suggested that salicylate inhibits binding of T(4) only to thyroxine-binding prealbumin (TBPA) and that T(3) is not bound to this protein. Using ultrafiltration techniques, we demonstrated binding of T(3) to TBPA. The affinity constant for T(3)-TBPA binding appears to be slightly greater than that for albumin-T(3) binding. While salicylate inhibits the binding of T(3) (and T(4)) to TBPA, it can be predicted that little change will be observed in the free T(3) (or free T(4)) without inhibition of thyroid hormone binding to thyroxine-binding globulin (TBG). Using a competitive-binding protein displacement technique, it has been shown that sodium salicylate, like diphenylhydantoin (DPH), inhibits the binding of T(3) and T(4) to TBG. The magnitude of the increase in free T(3) and free T(4) induced by salicylates suggests that interference with TBG binding is its major effect.Aspirin was administered orally to two normal subjects in quantities sufficient to obtain serum salicylate levels of 20-25 mg/100 ml. This resulted in a decrease of 20-30% in total serum T(3) and T(4) levels. This decrease in T(4) levels is similar in magnitude to that previously observed in subjects receiving DPH. Unlike what has been observed with DPH treatment, therapeutic salicylate levels are associated with increases of 50-75% in the unbound fraction of both T(3) and T(4) which persist throughout an 8-10 day treatment period.     

Ultrasensitive radioimmunoassay for direct determination of free triiodothyronine concentration in serum.

Free triiodothyronine (T3) in serum has been measured directly in dialysates of serum, using a wick chromatographic radioimmunoassay. Adequate sensitivity was attained by the use of [1251]T3 with very high specific activity (2,000 to 3,000 muCi/mug). Sera were dialysed against a Krebs-Ringer bicarbonate buffer modified so as to be similar to plasma water. Dialysis took place under carefully controlled circumstances. The influence on the equilibrium of total to free T3 of temperature, serum dilution, and dialysis time was studied. By the present method, free T3 in serum from groups of subjects including 20 men, 10 women taking oral contraceptives, and 20 women with normal menstrual cycles were identical, averaging 5.2 pg/ml. A chromatographic radioimmunoassay of total T3 using high specific activity [1251]T3 and very small test samples is also described.     

The influence of thyroxine on the serum free triiodothyronine concentration.

By means of measurement of the serum free triiodothyronine fraction (%FT3) by equilibrium dialysis, the influence of varying serum concentrations of thyroxine (T4) on %FT3 was studied in conditions associated with lack or excess of serum T4 and in serum enrichment with T4 in vitro. The enrichment of pooled serum with T4 caused significant increases of %FT3 with T4 was added to elevate its T4 content between 10 and 25 mug/dl. In 4 subjects with primary hypothyroidism treated with T3 (25 mug once daily for two weeks, twice daily thereafter), progressive increases of serum T3 were observed while their T4, free T4 and T3 fractions remained unchanged. Consequently, the serum free T3 and thyrotropin concentrations became normal after the serum T3 concentrations became hypernormal. When comparisons of the serum thyroid hormone levels were made between 6 subjects with T3 toxicosis and 8 with a usual variety of hyperthyroidism matched for serum T3 concentration, the former sub-group of hyperthyroidism showed significantly lower serum free T3 concentration (0-51 +/- 0.22 ng/dl versus 0.79 +/- 0-21 ng/dl, p less than 0.05). The amount of T4 in serum is considered to affect its free T3 concentration by virtue of sharing serum-binding proteins.     

Determination of triiodothyronine concentration in human serum

A simplified method has been described for the measurement of triiodothyronine (T3) in human serum. The sensitivity was sufficient for determinations on hypothyroid as well as normal and thyrotoxic sera. The values obtained have been in reasonable agreement with a double isotope derivative assay.The normal T3 concentration in human serum approximates 0.2 mug/100 ml; the mean +/-SD of 31 normal sera was 220 +/-27 ng/100 ml. Elevations were observed in sera from 40 patients with thyrotoxicosis (752 +/-282 ng/100 ml), and diminutions were found in sera from 10 hypothyroid patients (98+/-48 ng/100 ml).In rare instances thyrotoxicosis may be due to elevated serum T3 with normal thyroxine (T4) concentration. The incidence of this condition remains to be determined.In approximately half the cases with low serum T4 after (131)I therapy, the eumetabolic state may be maintained by normal or elevated T3 concentration.From these data and kinetic studies indicating a rapid turnover it may be inferred that T3 rather than T4 may be the more important hormone in health and in disease.     

Evaluation of serum triiodothyronine and adjusted triiodothyronine (free triiodothyronine index) in pregnancy.

We measured serum thyroxine (free and total), triiodothyronine (free and total), thyroxine-binding globulin, and triiodothyronine uptake by talc in 97 normal men and 50 pregnant women. Mean serum thyroxine and triiodothyronine concentrations were higher in the pregnant subjects (104 vs. 78 mug/liter and 1.69 vs. 1.30 mug/liter) because of a higher mean thyroxine-binding globulin concentration (70 vs. 38 mg/liter). Mean triiodothyronine uptake by talc was lower in the pregnant subjects (0.82 vs. 1.03). Mean free thyroxine concentrations were similar in the two groups, but mean free triiodothyronine concentrations were 10% lower in the pregnant subjects. Triiodothyronine uptake by talc and the diayzable thyroxine and triiodothyronine fractions were highly correlated (r = 0.85 and r = 0.82, P less than 0.001). Calculated free thyroxine index and free triiodothyronine index values (hyroxine and triiodothyronine indirectly adjusted, using triiodothyronine talc uptake to compensate for differences in thyroxine-binding globulin concentration), were statistically similar (84 vs. 82 and 1.38 vs. 1.34) in pregnant and male subjects. The results indicate that the total triiodothyronine concentration can be normalized on the basis of the triiodothyronine uptake by talc to correct for variations in thyroxine-binding globulin concentration.     

Total and free thyroxine and triiodothyronine: Measurement discrepancies,particularly in inpatients

ObjectiveWe compared the performance of tandem mass spectrometry versus immunoassay for measuring thyroid hormones in a diverse group of inpatients and outpatients.MethodsThyroxine (T4), triiodothyronine (T3), free thyroxine (FT4), and free triiodothyronine (FT3) were measured by liquid chromatography tandem mass spectrometry and immunoassay in 100 patients and the two assays were compared.ResultsT4 and T3 values measured by the two different assays correlated well with each other (r = 0.91–0.95). However, the correlation was less good at the extremes (r = 0.51–0.75). FT4 and FT3 concentrations measured by the two assays correlated less well with each other (r = 0.75 and 0.50 respectively). The studied analytes had poor inverse correlation with the log-transformed TSH values (r = -0.22 – 0.51) in the population as a whole. The strongest correlations were seen in the groups of outpatients (r = − 0.25−0.61). The weakest degree of correlation was noted in the inpatient group, with many correlations actually being positive.ConclusionThe worst between-assay correlation was demonstrated at low and high hormone concentrations, in the very concentration ranges where accurate assay performance is typically most clinically important. Based on the lesser susceptibility of mass spectrometry to interferences from conditions such as binding protein abnormalities, we speculate that mass spectrometry better reflects the clinical situation. In this mixed population of inpatients and outpatients, we also note failure of assays to conform to the anticipated inverse linear relationship between thyroid hormones and log-transformed TSH.     

Free triiodothyronine and free thyroxin in sera of pregnant women and subjects with congenitally increased or decreased thyroxin-binding globulin

Concentrations of free thyroxin (T4) and free triiodothyronine (T3) were measured in sera from pregnant women, in subjects with congenitally increased or decreased thyroxin-binding globulin (TBG), and in euthyroid controls. Measured free hormone concentrations were compared with calculated values for free hormone derived from concentrations of total T4, total T3, and binding proteins. Measured and calculated concentrations of free T4 and free T3 were below normal in the pregnant subjects but were normal in subjects with congenital increases of TBG. The low concentrations of free hormone in pregnancy are at variance with the euthyroid status of these subjects. Measured free T4 was normal, but concentrations of free T3 were less in the euthyroid congenitally low-TBG group.     

Diabetes mellitus in elderly persons.

The person who develops diabetes late in life needs carefull assessment of his abilities to assume the new self-care activities required by the advent of a chronic disease. Knowledge of both the aging process and the complications and management of diabetes is needed to assist him toward this goal. His commitment may have to be to a part of the whole plan, but to the elderly person, any participation will provide a measure of independence and control over his life.     

Enzyme immunoassay of free triiodothyronine in serum

In this new solid-phase antibody enzyme immunoassay for free triiodothyronine (FT3) in serum, beta-D-galactosidase conjugated to triiodothyronine is used. Results are uninfluenced by physiological concentrations of thyroxin-binding globulin or albumin. Results correlate well with those determined by equilibrium dialysis (r = 0.95). The mean CVs within and between assays were 6.1 and 9.5%, respectively. The measurable range of FT3 in serum is 0.7 to 26 ng/L; the normal reference interval is 1.9 to 8.9 ng/L. Concentrations of FT3 in serum of patients with hyperthyroidism were high; those of patients with hypothyroidism were within normal limits or low, and those of patients with congenitally decreased or increased TBG were within the normal range. In normal pregnant women, concentrations of FT3 as determined by radioimmunoassay correlated with those of albumin, declining as pregnancy progressed, but FT3 values determined by the proposed method or equilibrium dialysis were within the normal range and did not change during pregnancy.     

Dependence of free thyroxine estimates obtained with equilibrium tracer dialysis on the concentration of thyroxine-binding globulin.

Some equilibrium dialysis determinations of free thyroxine (T4) vary directly with thyroxine-binding globulin (TBG) concentration. This apparent TBG dependence has been limited to methods involving radiolabeled T4 added to the dialysis system (tracer dialysis). In this study we compared tracer dialysis with direct dialysis for determining free T4 and obtained the following results (mean +/- SD) for patients with hypothyroxinemia of nonthyroidal illness (23.8 +/- 10.7 vs 24.2 +/- 10.9 pmol/L, P greater than 0.8), patients with congenital TBG deficiency (11.4 +/- 2.2 vs 16.2 +/- 7.1 pmol/L, P greater than 0.05), normal control subjects (32.7 +/- 6.5 vs 18.5 +/- 5.8 pmol/L, P less than 0.001), and pregnant women (31.2 +/- 8.7 vs 12.1 +/- 2.6 pmol/L, P less than 0.001). Direct dialysis determinations were independent of TBG and total T4. Tracer determinations were greater than direct determinations in normals, a discrepancy that increased in pregnancy. Tracer determinations correlated significantly with total T4 and TBG concentrations (P less than 0.001). TBG and total T4 dependence in the tracer method was attributable to small overestimations of the free fraction of T4. Similar overestimations multiplied by increasing total T4 concentrations resulted in greater errors. Relative to results for normal sera, the tracer method overestimated free T4 when total T4 was increased and underestimated free T4 when total T4 was decreased.     

Serum free thyroxine concentrations in normal euthyroid subjects and ones with high serum thyroxine binding globulin concentration

Serum free thyroxine (fT4) was assayed by a commercial fT4 method in 30 normal euthyroid subjects, 19 pregnant females, 13 euthyroid subjects with high thyroxine binding globulin (TBG) and three with low or undetectable serum TBG concentration. In a number of these fT4 was also calculated on the basis of the application of the law of mass action to the binding situation. In states in which TBG was altered for congenital reasons both the experimentally determined and calculated fT4 were not significantly different from their respective means in the normal euthyroid population. Pregnant females had both lower experimental and theoretical free T4 concentrations. It is inferred from these data that TBG concentration per se is without effect on serum fT4 concentration.     

A two-step radioimmunoassay for free triiodothyronine in serum

Using a high-affinity solid-phase-bound antibody (Ka = 1.2 X 10(11) L/mol), we have standardized a two-step radioimmunoassay for free triiodothyronine (FT3) in serum, based on immunoextraction. The method was validated by comparison with an equilibrium-dialysis procedure (r = 0.96) involving RIA of T3 in the dialysate standardized with the same antibody and by a commercial (Liso-Phase, International-CIS) method. The two-step RIA could detect as little as 0.2 pg per milliliter. The mean CVs within and between assays were 9% and 12%, respectively. FT3 values measured in 30 normal adults ranged from 1.77 to 4.77 ng/L. Comparison with ratios of total T3 to thyroxin-binding globulin showed good agreement in normal subjects, pregnant women, and hypothyroid and hyperthyroid patients.     

Improved ultrafiltration method for simultaneous measurement of free thyroxin and free triiodothyronine in serum

We describe a new ultrafiltration method for measuring concentrations of free thyroxin (FT4) and free triiodothyronine (FT3). Serum containing [131I]T4 and [125I]T3 is diluted 10-fold in phosphate buffer (500 mmol/L, pH 7.4 at 37 degrees C), then passed through a YMT membrane in an Amicon MPS-1 centrifugal ultrafiltration device. Radioactive iodide and protein-bound thyronines are separated from FT4 and FT3 in the ultrafiltrate by use of a Sephadex G-25 microcolumn. In normal controls, the fraction of T4 that passes into the ultrafiltrate is 0.021 +/- 0.006% (mean +/- SD), FT4 is 19.6 +/- 6.5 ng/L, the fraction of T3 that passes into the ultrafiltrate is 0.18 +/- 0.5%, and the FT3 is 2.03 +/- 0.50 ng/L. Intra-assay precision (CV) is 5.4% for FT4 and 4.2% for FT3; the respective interassay CVs are 10.1% and 8.0%. In various groups of patients with thyroid disease and other conditions that influence serum T4 and T3 concentrations, FT4 by ultrafiltration correlated with FT4 measured by equilibrium dialysis (r = 0.84, p less than 0.001) and FT3 by ultrafiltration correlated with FT3 measured by the Diagnostic Products RIA kit (r = 0.87, p less than 0.001). The accuracy, reproducibility, speed, and simplicity of the ultrafiltration method compared favorably with the more cumbersome method of equilibrium dialysis.