Value of free thyroxine (FT4), free triiodothyronine (FT3), and sensitive thyrotropin (TSH) assay in the assessment of optimal thyroxine therapy

We describe a sensitive thyrotropin (TSH) assay in the evaluation of thyroxine replacement therapy. Patients taking varying amounts of L-thyroxine replacement doses were studied using a thyrotropin-releasing hormone (TRH) test and a sensitive TSH assay as the indices of optimal replacement therapy. There were no differences in the mean thyroxine doses of those patients who had a normal TRH response and those who had a flat response. Similarly there were no significant differences in the serum free thyroxine (FT4), free triiodothyronine (FT3), or total triiodothyronine (TT3) levels between these two groups. The patients in both groups had normal basal serum TSH values as measured by a standard, HTSH RIABEAD (Abbott) method. However, serum TSH values, as measured by a sensitive TSH3 MAIACLONE (Serono) immunoradiometric method, were subnormal in all patients with flat TRH responses. The TSH (Serono) assay provided the best single blood test of optimal thyroxine replacement.     

Reference intervals from birth to adulthood for serum thyroxine (T4), triiodothyronine (T3), free T3, free T4, thyroxine binding globulin (TBG) and thyrotropin (TSH).

Disorders in thyroid function can impair normal development in children. Therefore it was our aim to establish reference intervals for serum triiodothyronine (T3), free T3 (fT3), thyroxine (T4), free T4 (fT4), thyroxine binding globulin (TBG) and thyrotropin (TSH) which are applicable from birth to adulthood by using the non-isotopic automated chemiluminescence immunoassay system, Immulite (DPC Los Angeles, USA). Serum samples from 762 euthyroid newborns, children and adolescents (369 female, 393 male; age 1 day to 19 years) were examined; of these, 381 were classified as pubertal. Due to non-normal distribution, the 2.5th, 50th and 97.5th percentiles (the central 95% interval) were calculated for each group. The median concentrations of T4, fT4 and TSH were up to 3.2-fold higher during the first 2 weeks, while T4 increased during the first month of life. The concentrations in all age groups showed no sex differences. From 1 year onwards, the concentration of all parameters tended to decrease until adult age, with the exception of TBG which increased by >60% (p<0.02) and reached a maximum at approximately 5 years of age. The findings underscore the fact that thyroid hormones are not associated with sexual development, except for TBG, which decreased slightly (p<0.04) between Tanner stages 1 and 5. However, the reference intervals established here demonstrate that marked changes occur in concentrations of thyroid hormones after the neonatal period. Our findings complement these of earlier studies. The developed reference intervals can be used to assess the thyroid status of patients, particularly if the measurements are done on the Immulite/Immulite 2000 system.     

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.     

Semi-automated radioimmunoassays for total serum thyroxine and triiodothyronine

Single stage semi-automated radioimmunoassays for total serum thyroxine (T4) and triiodothyronine (T3) are described which employ an automatic pipetting station, automatic gamma counter, and a programmable calculator with paper tape reader and printing facility. Both assays require only a small volume of unextracted serum, and are specific and sensitive. Their sample capacity, precision, speed, and cost are comparable with the measurement of serum protein-bound iodine. Both assays therefore have significant advantages over previous methods for the assessment of thyroid function in the diagnostic laboratory.A simple method of automating the calculation of results is described, which is applicable to any radioimmunoassay in which the standard curve is approximately linear on a plot of the free/bound fraction against the antigen concentration. In addition, a general method is reported which reveals the relative contributions of intrinsic, systematic, and random error to radio-immunoassay precision.     

Estimation of thyroxine and triiodothyronine distribution and of the conversion rate of thyroxine to triiodothyronine in man.

Studies on peripheral metabolism of simultaneously administered 125-I-labeled L-thyroxine ([125-I]T4) and 131-I labeled L-trilodothyronine ([131-I]T3) were performed in five normal subjects, in four patients with untreated hypothyroidism, and in 3 hypothyroid patients made euthyroid by the administration of T4. The fractional turnover rate (lambda 03) of thyroid hormones irreversibly leaving the site of degradation and the volumes of pool 1 (serum V1) of pool (interstitial fluid, V2), and of pool 3 (all tissues, V3)were obtained by using a three-compartment analysis. In addition to the turnover studies, the ratios for the in vivo T4 to T3 conversion were determined by paper chromatographic study in sera obtained 4, 7, and 10 daysafter the injection. The rate (K12) of the extrathyroidal conversion of T4 to T3 was also estimated by the compartment analysis. The T3 distribution volume (V3) of pool 3, in which T3 is utilized and degraded, was about 60% of totaldistribution volume (V=V1+V2+V3) in normal subjects, whereas only about 25% of the extrathyroidal T4 pool was in the intracellular compartment, indicating that T3 is predominantly an intracellular hormone..     

Evaluation of a new enzyme immunoassay system for free thyroxine (Enzymun-Test FT4)

Free thyroxine (FT4) represents the metabolically active fraction of the circulating thyroid hormone thyroxine (T4). In this paper the results of the evaluation of a newly developed FT4 assay are reported. This assay system is based on an enzyme-labelled one-step immunoassay. The within-run imprecision, checked using serum pools and several commercial reference materials, showed a coefficient of variation (CV) of between 0.8 and 9.8%, depending on the reference material used. The between-run imprecision showed a CV of between 1.0 and 13.2%. Accuracy experiments yielded values between 80 and 116%. When the new FT4 was compared with the calculation of an index for free thyroxine (FT4I; derived from either the ratio of T4/thyroxine binding coefficient of from T4/thyroxine binding globulin) in a number of samples in the hypo-, eu- and hyperthyroid range, a good correlation was obtained. The same was true when the new FT4 assay was compared with a widely used two-step radioimmunoassay (y = -0.146 + 0.943 x). In euthyroid subjects the measured FT4 concentration was 10.3-25.8 pmol/l. No effect was evident when the influence of EDTA and citrate was investigated, whereas addition of heparin led to an increase in FT4 concentration of about 12 to 15%. Investigation of the possible influence of a large number of drugs showed that probenezid, carbamazepine and furosemide led to an increase in the measured FT4 concentration. Dialysis increased the FT4 concentration, as measured in patients before and after haemodialysis. No effect of alteration in protein concentration and/or protein distribution of FT4 concentration could be detected. In pregnancy, FT4 values were within the normal range.(ABSTRACT TRUNCATED AT 250 WORDS)     

A critical evaluation of separation methods in radiommunoassay for total triiodothyronine and thyroxine in unextracted human serum

Methods for separating free and antibody-bound hormone in radioimmunoassays for total triiodothyronine (T-3) and thyroxine (T-4) in unextracted human serum are evaluated. For T-3 assay, a simplified second antibody technique has significant advantages over other methods and gives a mean interassay coefficient of variation of 7.2% over a wide range of values. For T-4 assay, polyethylene glycol is the method of choice and has a mean interassay coefficent of variation of 4.7%. By adding the separating agents initially, the assays are readily semi-automated and may be completed within a working day.     

Evaluation of five methods for determining low-density lipoprotein cholesterol (LDL-C) in hemodialysis patients(1)

Objectives: Current recommendations for the management of dyslipidemia are largely based on the concentration of LDL-C. Most clinical laboratories estimate the concentration of LDL-C by the recommended routine method, the equation of Friedewald, in specimens from fasting subjects and with TG concentrations < 4.52 mmol/L. Because of the limitations of the Friedewald calculation, direct methods for an accurate quantification of LDL-C are needed.

Design and Methods: In the present study we evaluated the accuracy of the following 5 different procedures for LDL-C in 98 patients on hemodialysis: the Friedewald equation, where LDL-C is calculated from HDL-C, measured either by the precipitation procedure with dextran sulfate-Mg²⁺ (Method 1), or by a direct HDL-C assay (Method 2), the Direct LDL™ assay (Method 3), the homogeneous N-geneous™ LDL assay (Method 4) and the calculated LDL-C values deriving from the ApoB based equation: 0.41TC - 0.32TG + 1.70ApoB - 0.27, (Clin Chem 1997;43:808–815) (Method 5).

Results: All five LDL-C methods were found to be in good agreement with ultracentrifugation/dextran sulfate-Mg²⁺ precipitation with the coefficients of correlation of the assays to ranging between 0.93–0.95. However, significant differences in the mean values and biases vs. the reference method were observed. The Friedewald equation and the Direct assay were less affected by high LDL-C levels, and they presented higher sensitivity and higher negative predictive value. The N-geneous assay and the ApoB derived calculation were less affected by high triglyceride levels, and they presented higher specificity and higher positive predictive value. At the diagnostic LDL-C level of 3.37 mmol/L, both Friedewald calculations correctly classified 82/92 patients; Direct assay 86/98; N-geneous assay 88/98; and ApoB derived calculation 88/98. At the diagnostic LDL-C level of 2.98 mmol/L, Friedewald calculations (Method 1 and Method 2) correctly classified 82/92 and 81/92 patients, respectively; Direct assay (LDL-3) 87/98; N-geneous assay (LDL-4) 91/98; and ApoB derived calculation (LDL-5) 91/98.

Conclusions: Among hemodialysis patients, who commonly present “average” LDL-C concentrations and high TG levels, the N-geneous assay and the apoB derived calculation seem to yield more acceptable results for the estimation of LDL-C.     

Production and storage of (125-I) thyroxine and (125-I) triiodothyronine of high specific activity.

High specific activity [125-I] triiodothyronine and [125-I] thyroxine have been produced regularly by the chloramine T radioiodination method. Simultaneous production of [125-I] triiodothyronine and [125-I] thyroxine is usual when diiodothyronine or triiodothyronine are employed as the starting materials. Specific activities reached vary with the starting compound (diiodothyronine, triiodothyronine, thyroxine) used, as both substitution and, less readily, exchange of iodine atoms take place. Starting with diiodothyronine specific activities of approximately 2400 and 5200 Ci/g were achieved for [125-I]triidothyronine and [125I] thyroxine, respectively, and, similarly, specific activities of approximately 1200 and 4000 Ci/g for [125I] triiodothyronine and [125I]-thyroxine, respectively, were reached when triiodothyronine was the starting material. [125-I] Triiodoacetic acid and [125I] tetraiodoacetic acid have been produced in the same way from triiodoacetic acid. By column chromatography on Sephadex G-25 (fine), eluting with alkaline phosphate buffer, good separation of the radioiodinated products has been readily achieved. Studies on the stability of the radioiodinated hormones showed that 50% methanol, ethanol, propanol and propylene glycol were all equivalent as preserving agents and, further, that the stability of the radioiodinated hormones was linearly related to the concentration of these preserving agents.     

Conversion of Thyroxine (T4) to triiodothyronine (T3) in athyreotic human subjects

Studies of the possibility that thyroxine (T4) is converted to 3.5,3′-triiodo-L-thyronine (T3) in the extrathyroidal tissues in man have been conducted in 13 patients, all but two of whom were athyreotic or hypothyroid, and all of whom were receiving at least physiological replacement doses of synthetic sodium-L-thyroxine.