Determination Of Serum Thyroxine Research Articles

Isotope dilution-liquid chromatography/electrospray ionization-tandem mass spectrometry for the determination of serum thyroxine as a potential reference method.

A new candidate reference method is presented for the determination of thyroxine in serum. The method is based on isotope dilution-liquid chromatography/tandem mass spectrometry using electrospray for ionization. The internal standard used was 13C6-thyroxine, sample pretreatment consisted of protein precipitation and a two-step liquid/liquid extraction procedure, HPLC was performed on a C-18 column with an eluent containing methanol/water/formic acid (60:40:0.1, by volume), and finally thyroxine and its isotopically labeled analogue were measured in the selected reaction monitoring mode for the transitions m/z 777.7--> 731.7 and m/z 783.7--> 737.7, respectively. The detection limit for thyroxine was 6 pg, the within-run coefficient of variation was 1.1%. The samples were measured in six-fold: in duplicate on three independent days. The mean overall coefficient of variation of the method was 1.6%. The new method was evaluated by measuring nine control sera previously determined by an existing ID-GC/MS method. The differences between the results of the two methods ranged from-1.6% to +3.3%, with a mean of +0.2%.

Development of a new method for the determination of thyroxine in serum based on isotope dilution gas chromatography mass spectrometry.

A new gas chromatographic/mass spectrometric method in combination with isotope dilution for the determination of thyroxine in serum is described. Special attention was paid to the methylation step of thyroxine, which was investigated using methanolic HCl, dimethylformamide/dimethylacetal and diazomethane, the latter giving the best results in terms of reproducible isotope ratios. For internal standardization, (13C6)-thyroxine was dissolved in fraction V human albumin solution (70 g l-1). The internal standard-in-albumin solution was mixed with known amounts of thyroxine standard, dissolved in 0.05 M Na2HPO4 buffer at pH 11.6, to give isotope ratios of 0.75, 1.00 and 1.25. The same internal standard solution was also used for isotope dilution of the unknown serum samples. The volume of serum was adapted to give a 1:1 isotope ratio. Sample pretreatment consisted of protein precipitation and a two-step liquid/liquid extraction procedure. After methylation of unlabelled and labelled thyroxine with diazomethane and perfluoroacylation with pentafluoropropionic anhydride and heptafluorobutyric anhydride, respectively, mass spectrometric monitoring was done at m/z 951/957 and 1001/1007. Quantitative determination of thyroxine in five serum samples in duplicate, during three consecutive days, showed a mean overall imprecision of 1.0% and a deviation of +0.4% from the target value as determined by a definitive method.

Determination of albumin-bound thyroxin in serum

Determination of albumin-bound thyroxin in serum

Homogeneous time-resolved fluoroimmunoassay of thyroxin in serum.

We describe a rapid, simple nonseparation fluoroimmunoassay for determination of thyroxin in serum. The assay is based on the labeling of thyroxin directly with a fluorescent europium chelate, the fluorescence of which is quenched on binding to an antithyroxin antibody. With the assay buffer we used, maximum quenching is 90%. The rapid achievement of equilibrium in the assay solution, regardless of the sequence of reagent additions, allows fast measurement of thyroxin. Precision was good (CV less than 5%) within the clinical range for total thyroxin (50-300 nmol/L), and results correlated well with those by a commercial radioimmunoassay.

Determination of free thyroxin in serum by ultrafiltration: validation of a method and preliminary results.

We describe the determination of concentrations of free thyroxin in serum by ultrafiltration based on the Amicon micropartition system. The assay is straightforward and requires about 4 h for 25 samples. Our results demonstrate a correlation coefficient of 0.81 compared with equilibrium dialysis, and of 0.88 compared with radioimmunoassay. For sera from 49 normal, 25 hyperthyroid, 24 hypothyroid, 50 euthyroid sick, and 13 pregnant subjects, the means (and SD) were respectively: 19.3 (4.9), 40.1 (13.6), 5.0 (2.9), 25.9 (10.5), and 16.1 (3.2) ng/L. The interassay CV was 9.9% and the intra-assay CV 7.2%. We conclude that this procedure is useful in the diagnosis of thyroid disease and offers advantages of simplicity, rapidity, and economy.

Enzyme immunoassay of thyroxin with a centrifugal analyzer.

We have applied a homogeneous enzyme immunoassay for determination of thyroxin in serum to the "Cobas Bio" centrifugal analyzer. To unbind thyroxin from its protein complex, serum is treated for 20 min with a solution of NaOH containing "Lipex," an agent for sequestering free fatty acids. The immunoenzymic reaction is then automatically performed by the analyzer at 37 degrees C. To 20 microL of sample mixture is added 125 microL of reagent (thyroxin antibodies and NAD+) and this mixture is incubated for 10 s. Then 25 microL of start reagent (enzyme-thyroxin conjugate and malate substrate) is added and the change in absorbance is monitored at 340 nm. The standard curve is linear up to at least 200 micrograms of thyroxin per liter. Within-assay precision (CV) varied from 1.1 to 2.9%, between-assay precision from 3.1 to 7.8%. Analytical recovery of thyroxin was complete. The deviation of control samples from target values ranged from -2.1% to 7.0%. Interference by hemoglobin or bilirubin is negligible. Results compare favorably with those by radioimmunoassay.

Evaluation of Enzyme Immunoassays for Determination of Thyroxine (EMIT, ENZYMUN) and of Thyroxine Binding Index

An evaluation of enzyme immunoassays for determination of thyroxine in serum (EMIT ABA thyroxine assay, Syva Corp., ENZYMUN assay thyroxine, Boehringer Mannheim) and of thyroxine binding index (ENZYMUN assay TBI1), Boehringer Mannheim) is presented. The precision of the enzyme immunoassays was adequate (coefficients of variation ranged from day to day with EMIT from 3--11% and with ENZYMUN from 4--11%). Both assays are specific and easy to perform. About 20 unknown samples can be analyzed in duplicate by EMIT within 60 minutes and by ENZYMUN within 250 minutes. A comparison of the results obtained by enzyme immunoassays and radioimmunoassay in a series of about 100 patients showed a good correlation between both methods. The precision of the ENZYMUN TBI assay was adequate (coefficient of variation from day to day 4.9%) and the thyroxine/TBI-ratio correlated well with the thyroxine/TBG-ratio.

Determination of thyroxine in serum by gammacoat T4 (author's transl)

Determination of thyroxine in serum by gammacoat T4 (author's transl)

Determination of serum thyroxine in two species of toads: Variation with season

Thyroxine concentrations were compared in two species of toads, viz., the green toad, Bufo viridis, and the common toad, B. bufo. Thyroxine was determined by means of a competitive protein-binding technique which had a detection level below 0.1 ng. The method is described and its applications are discussed. In prebreeding B. viridis, thyroxine levels were 13.6 ± 0.9 ng/ml of serum in females and 10.6 ± 0.8 ng/ml in males. In early autumn the concentration had decreased to 5.0 ± 0.4 ng/ml. B. bufo had 3.5 ± 0.3 ng/ml of serum in June and remained at that level until October. Common toads kept for the winter in a refrigerator at 5° and sacrificed in December and February had 1.7 ± 0.1 ng of thyroxine/ml of serum.

Determination of free thyroxine in serum by radioimmunoassay.

We describe a sensitive and specific radioimmunoassay for free thyroxine, directly determined in the diffusate obtained from equilibrium dialysis of serum. The intra- and interassay precisions were 7.5 +/- 0.27 and 9.1 +/- 0.58 ng/liter (mean +/- SD), respectively. The free thyroxine concentrations of normal persons and patients with thyroid disease were as follows: normal, 7.6 +/- 1.7; euthyroid patients receiving phenytoin, 6.9 +/- 1.4; hypothyroidism, 2.9 +/- 1.3; and hyperthyroidism, 32.3 +/- 12.3 ng/liter (mean +/- SD).

Evaluation of sodium l-thyroxine (T 4) requirement in replacement therapy of hypothyroidism

Sodium-L-thyroxine (T4) was utilized in the treatment of 15 pediatric patients with hypothyroidism. Adequacy of replacement therapy was confirmed by clinical evaluation in conjunction with determination of serum thyroxine, tri-odothyronine, and thyrotropin concentrations. Daily dose of thyroxine capable of including clinical and biochemical euthyroidism ranged from 2.5 to 5 mug/kg of body weight with a mean of 3.5 +/- 0.3 mug in the 4- to 17-year-old age group. Two infants with congenital hypothyroidism diagnosese observations imply that physiologic requirements of thyroxine are distinctly lower than previously recommended dosages. Further studies to establish more precise therapeutic guidelines are needed.

Determination of Serum Thyroxine after Dissociation from Thyroxine-Binding Globulin in Alkaline Solution and Adsorption on Dextran-Coated Charcoal

A competitive protein-binding procedure for determining serum thyroxine is described in which thyroxine is described in which thyroxine is first dissociated from serum thyroxine-binding globulins in an alkaline solution. After binding equilibration, the unbound thyroxine is separated by dextran-coated charcoal. The procedure takes only 100 mul of serum, requires no evaporation, and is sensitive and reproducible. Results by this procedure correlate well with those for a generally accepted method.

The distribution of Mn (super 2+) and Fe (super 2+) between ilmenites and magnetites in igneous rocks

OBJECTIVES--To determine whether thyroid dysfunction is found with increased frequency in patients with rheumatoid arthritis (RA). METHODS--A controlled prospective survey was conducted on a cohort of patients with RA derived from a hospital clinic and a private surburban rheumatology practice. A control group with similar demographic features was generated from the same sources and included subjects with either osteoarthritis or fibromyalgia. Consecutive patients were evaluated over a six month period. The evaluation included a complete history and physical examination, and determination of serum thyroxine, free thyroxine, triiodothyronine, thyroid stimulating hormone (IRMA), antinuclear antibodies, and rheumatoid factor. RESULTS--Of the 91 women with RA evaluated, 29 (30%) had evidence of thyroid dysfunction compared with 10 (11%) of 93 controls. The excess thyroid dysfunction is due to either hypothyroidism or Hashimoto9s thyroiditis and was independent of age, increasing duration of disease, rheumatoid factor, and antinuclear antibodies. CONCLUSIONS--Thyroid dysfunction is seen at least three times more often in women with RA than in women with similar demographic features with non-inflammatory rheumatic diseases such as osteoarthritis and fibromyalgia.

Laboratory Examination of the Thyroid

The in vitro tests of thyroid function measure serum levels of the thyroid hormones and are widely used for both screening and diagnosis. Of these, determination of serum thyroxine by displacement, known as the Murphy-Pattee method, is virtually free of interference and therefore is probably the best single test of thyroid activity.

Comparative Study of Thyroxine Assays Employing Kit Radio-Ligand Reagents

A comparative study of some ‘kit’ radiometric procedures for the determination of thyroxine in serum is reported. Four manufacturers' competitive-binding reagents and methods—three incorporating prior extraction and one employing column elution—are examined. Considerable discrepancies exist between the manufacturers' ‘standards’ which, in the main, account for the differences in results obtained by the different methods. Other deficiencies include susceptibility to metal ion interference, variation (up to 8%) in thyroxine extraction and non-specific, i.e. ‘mimic T-4’, measurement. Radio-ligand reagent ‘kits’ need to be critically assessed before being used routinely. Separation of hypo-and hyperthyroid from euthyroid patients is achieved as well with some competitive protein-binding T-4 methods as with serum PBI.

Simple Method for Determining the Concentration of Serum Thyroxine by Competitive Displacement Analysis

SummaryA simplified method for the determination of serum thyroxine is described. The mathematical formulation of the standard curve required for the competitive displacement analysis permits an evaluation of the serum thyroxine concentration with the use of two standard values instead of measuring the full curve. The values calculated from the formula show a very close correlation with those obtained by comparison with the standard curve.

Determination of Organic Iodine Compounds in Serum

Abstract Unidentified substances in acetic acid column eluates, obtained in the determination of serum thyroxine by column chromatography with the anion-exchange resin Dowex-1, prevented the direct determination of thyroxine by interference with the iodidecatalyzed ceric-arsenite reaction. Pretreatment of the eluates with Br2 or Cl2 eliminated the interference and permitted the direct assay of thyroxine by the ceric-arsenite system without incineration or wet digestion. Pretreatment with Br2 or Cl2 also significantly enhanced the catalytic activity of thyroxine, rendering its iodine nearly equivalent to inorganic iodide in the ceric-arsenite system. This nonincineration thyroxine assay is significantly more specific than methods that employ wet or dry ashing, because many organic iodine compounds used in diagnosis and therapy are not measured. A comparison of the nonincineration technic with the alkaline dry-ash method on 110 random serums revealed a mean difference of 0.13 µg./100 ml. (X = 4.5 µg./ 100 ml.), with the nonincineration technic yielding the higher result. This difference was found to be statistically significant (p< 0.001).

Determination of serum thyroxine. The diagnostic value in thyroid diseases.

Determination of serum thyroxine. The diagnostic value in thyroid diseases.

A new and simple method for the determination of thyroxine in serum.

A new and simple method for measurement of serum T4 is introduced in which a modified 131I-T3 resin sponge uptake is used. Unlike the conventional 131I-T3 resin sponge uptake, this new method can measure T4 concentration in the serum without regard to the amount of T4 binding protein in the serum sample. After 2 ml of the serum sample has been deproteinized, the standard stock serum is added to the free T4. Then the 131I-T3 resin sponge uptake method is applied to this mixture. The T4 content of the serum sample is measured using the standard calibration curve of 131I-T3 resin uptake values obtained using various concentrations of a T4 solution. The values for T4 iodine (μg/100 ml) in various groups of diseases were: 1) hypothyroidism, 2.3 ±0.70 (mean ±sd); 2) euthyroid subjects, 5.7 ±1.08; 3) hyperthyroidism, 10.9 ±2.30; 4) pregnancy, 7.0 ±1.31; and 5) nephrosis, 2.3 ±0.94. Satisfactory reproducibility was observed by the duplicate determinations. These results proved that this method could be used as a ...

A new simple method for the determination of thyroxine in serum. (2)

A new simple method for the determination of thyroxine in serum. (2)