Free Thyroxine In Human Serum Research Articles

Description and validation of an equilibrium dialysis ID-LC-MS/MS candidate reference measurement procedure for free thyroxine in human serum.

Free thyroxine (FT4) in serum is routinely measured in clinical practice to diagnose and monitor thyroid disease. Due to its concentration in picomolar range and the delicate equilibrium of free and protein-bound T4, accurate measurement is challenging. As a consequence, large inter-method differences in FT4 results exists. Optimal method design and standardization of the FT4 measurement is therefore necessary. The IFCC Working Group for Standardization of Thyroid Function Tests proposed a reference system with a conventional reference measurement procedure (cRMP) for FT4 in serum. In this study, wedescribe our FT4 candidate cRMP and its validation in clinical samples. This candidate cRMP is based on equilibrium dialysis (ED) combined with determination of T4 with an isotope-dilution liquid chromatography tandem mass-spectrometry (ID-LC-MS/MS) procedure and was developed according to the endorsed conventions. Its accuracy, reliability, and comparability was investigated using human sera. It was shown that the candidate cRMP adhered to the conventions and its accuracy, precision, and robustness were adequate in serum of healthy volunteers. Our candidate cRMP measures FT4 accurately and performs well in serum matrix.

Development of an equilibrium dialysis ID-UPLC-MS/MS candidate reference measurement procedure for free thyroxine in human serum

BackgroundAccurate and reliable measurement of human serum free thyroxine (FT4) is critical for the diagnosis and treatment of thyroid diseases. However, concerns have been raised regarding the performance of FT4 measurements in patient care. Centers for Disease Control and Prevention Clinical Standardization Programs (CDC-CSP) address these concerns by creating a FT4 standardization program to standardize FT4 measurements. The study aims to develop a highly accurate and precise candidate Reference Measurement Procedure (cRMP), as one key component of CDC-CSP, for standardization of FT4 measurements. MethodsSerum FT4 was separated from protein-bound thyroxine with equilibrium dialysis (ED) following the recommended conditions in the Clinical and Laboratory Standards Institute C45-A guideline and the published RMP [20,21,23]. FT4 in dialysate was directly quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. Gravimetric measurements of specimens and calibrator solutions, calibrator bracketing, isotope dilution, enhanced chromatographic resolution, and T4 specific mass transitions were used to ensure the accuracy, precision, and specificity of the cRMP. ResultsThe described cRMP agreed well with the established RMP and two other cRMPs in an interlaboratory comparison study. The mean biases of each method to the overall laboratory mean were within ±2.5%. The intra-day, inter-day, and total imprecision for the cRMP were within 4.4%. The limit of detection was 0.90 pmol/L, which was sufficiently sensitive to determine FT4 for patients with hypothyroidism. The structural analogs of T4 and endogenous components in dialysate did not interfere with the measurements. ConclusionOur ED-LC-MS/MS cRMP provides high accuracy, precision, specificity, and sensitivity for FT4 measurement. The cRMP can serve as a higher-order standard for establishing measurement traceability and provide an accuracy base for the standardization of FT4 assays.

Development of solid support using protein A for the measurement of free thyroxine in human serum

Development of solid support using protein A for the measurement of free thyroxine in human serum

Magnetic particle-based chemiluminescence enzyme immunoassay for free thyroxine in human serum

A magnetic particles-based chemiluminescence enzyme immunoassay with high sensitivity, specificity, rapidity, and reproducibility was developed for the determination of free thyroxine in human serum. A competitive assay has been proposed with horseradish peroxidase labeled thyroxine analog. The immunomagnetic particles coated with anti-fluorescein isothiocyanate antibody was used as dispersed solid phase and separation means for the immunoassay. Experimental conditions, such as temperature, the volume of magnetic particles and substrate, incubation time, dilution ratio and other relevant variables upon the immunoassay have been examined and optimized. The proposed method exhibited high performance which the linear range was 1.59–122 pmol L −1 and the detection limit was 0.25 pmol L −1. A coefficient of variance of less than 15% was obtained for both intra-assay and inter-assay precision. The present method has been successfully applied to the analysis of free thyroxine in human serum. The diagnostic accordance rate of the method for normal serum, hyperthyroidism and hypothyroidism are satisfactory. Good correlations were obtained between the results by the proposed method and the commercial radioimmunoassay kit. The present method exhibits good potential in the fabrication of FT 4 diagnostic kits which could be used in the clinical analysis and facilitated the development of automated operation systems in the clinical practice.

Development of a highly sensitive and selective microplate chemiluminescence enzyme immunoassay for the determination of free thyroxine in human serum

A microplate chemiluminescence enzyme immunoassay (CLEIA) with high sensitivity, selectivity and reproducibility was developed for the determination of free thyroxine (FT4) in human serum. A competitive assay has been utilized with horseradish peroxidase (HRP) labeled thyroxine analog in the chemiluminescence (CL) detection. The CL signal produced by the emission of photons from luminol was directly proportional to the amount of analyte. The linear range was 0.45-7.5 ng dL(-1 )and the detection limit was 0.09 ng dL(-1). Experimental conditions, such as temperature, pH, incubation time, titration level and other relevant variables upon the CL signal have been examined and optimized. A coefficient of variance of less than 16% was obtained for intra- and inter-assay precision. The present method has been successfully applied to the analysis of FT4 in human serum. The positive and negative coincidence ratios are satisfactory. Good correlations were obtained between the results by the proposed method and radioimmunoassay (RIA), as well as a Bayer ACS-180SE detection system.

Deprecating the diagnostic significance of free thyroxine levels in human serum.

Prime diagnostic importance has been postulated for the measurement of free thyroxine in human serum. During the past decade various innovative and routinely applicable techniques have been developed for its assay, and claims for their superior performance in clinical practice have subsequently been made. However, in our opinion, based on current literature concerning thyroxine metabolism together with basic information about the physicochemical characteristics that govern its intravascular distribution, it seems appropriate to indicate caution against overestimating the diagnostic sensitivity attributed to free thyroxine measurement irrespective of assay methodology and its conceptual elegance.

Effect of free fatty acids on the concentration of free thyroxine in human serum: the role of albumin.

The concentration of FFA in normal human plasma in vivo generally ranges between 0.2 and 0.7 meq/liter; slightly higher concentrations have occasionally been reported in patients who are seriously ill. To determine whether such FFA concentrations may increase the concentration of free T4 in serum, we added increasing amounts of oleic acid to pooled normal human serum (with known FFA content) and measured free T4 by equilibrium dialysis. Total FFA up to 3 meq/liter in normal serum, representing an FFA to albumin molar ratio of about 5:1, had little or no effect on the free T4 concentration, while higher FFA concentrations progressively increased free T4. This same molar ratio of FFA to albumin had to be exceeded to cause a significant increase in the free T4 concentration in diluted serum and in serum from patients with nonthyroid illness. Serum from which more than 95% of the albumin had been removed by chromatography with Affi-Gel blue was much more sensitive to the effects of FFA on free T4. This enhanced sensitivity was reversed by readdition of albumin to the serum, and the addition of albumin to normal serum resulted in diminished effects of FFA on free T4. These results indicate the following: physiological concentrations of FFA do not significantly increase the free T4 concentration in normal human serum; when FFA reach supraphysiological concentrations in serum (in vitro) and the higher affinity FFA-binding sites on albumin become saturated (apparently at an FFA to albumin molar ratio of approximately 5:1), the excess FFA interact with other serum proteins, including thyroid hormone-binding globulin, and thereby increase the free T4 concentration; the concentration of albumin (or other FFA binders) must be considered when evaluating the observed effects of FFA. To explore the relevance of these findings to the hypothesis that FFA may inhibit the binding of T4 to plasma proteins in patients with nonthyroid illness, we measured plasma FFA concentrations in 11 severely ill patients hospitalized in the intensive care unit. We found a mean plasma FFA concentration of 0.45 +/- 0.11 (+/- SEM) mEq/liter and a mean serum albumin concentration of 2.39 +/- 0.29 g/dl in these patients. Their mean plasma FFA to albumin molar ratio was 1.53 +/- 0.41. Since the FFA to albumin molar ratio must exceed about approximately 5:1 before a significant increase in the serum free T4 concentration occurs, these results suggest that FFA do not commonly influence the circulating free T4 concentration in vivo, even in severely ill patients.

Determination of free thyroxine in human serum by gel bead microdialysis: R. Lavole, J. Guerin, G. Hamel, A. Joncas, S. Belanger, J.C. Forest. Service de biochimie, hopital Saint-Francois-d'Assise, Quebec, G1L 3L5

Determination of free thyroxine in human serum by gel bead microdialysis: R. Lavole, J. Guerin, G. Hamel, A. Joncas, S. Belanger, J.C. Forest. Service de biochimie, hopital Saint-Francois-d'Assise, Quebec, G1L 3L5

A new radioimmunological method for determining free thyroxine

A new method for determining free thyroxine in human serum was evaluated. It is a radioimmunological direct assay method using an anti-thyroxine antiserum which is very specific and bound to a solid-phase. To determine the accuracy of the method, we measured ‘with-in-assay’ reproducibility to have a CV% maximum of 2.9, and ‘between-assay’ reproducibility (for three sera with three different FT4 levels) to have a mean CV% of 8.2; the reproducibility of the standard curves in 12 different assays showed a maximum CV% of 9.2. FT4 levels were also evaluated in 167 subjects: in 55 normal subjects a mean FT4 value equal to 13.99 ng/l (range 8.8–19.1) was obtained; in 25 euthyroid subjects with high TBG and in 5 with low TBG normal values were obtained in 97% of the cases, whilst FTI values were within normal limits in only 77%; in 25 euthyroid patients with goitre, FT4 values were always within normal limits, in contrast with TT4 and FTI values (92%); in 25 patients with hyperactive ‘hot’ thyroid nodules, values which were on average higher (15.2 ng/l) than the range were obtained in 24% of the cases, whilst TT4 and FTI were higher than the range in 12% of the cases; in hyperthyroid (no.=24) and hypothyroid patients (no.=16), FT4 values were not within the normal range. The assay method was compared with another commercial method (radioimmunological, after separation of bound and free fractions by chromatography on Sephadex). There was a highly satisfactory correlation between the two methods (y=4.12+0.96x, r=0.982) in 123 samples, although a tendency was observed for the new method to show higher values than the older. We conclude by stating that the method studies is convenient and has acceptable accuracy and clinico-diagnostic reliability.

Microencapsulated antibodies in radioimmunoassay. II. Determination of free thyroxine.

We describe a method for directly measuring free thyroxine in human serum. Antibody to thyroxine was encapsulated within a semipermeable nylon membrane that excludes substances of relative molecular mass greater than 20,000. The microencapsulated antibody was then presaturated with [125I]thyroxine. Serum incubated with the microcapsules initiated a displacement reaction between free thyroxine and [125I]thyroxine bound to the antibody. The displaced thyroxine was separated from the bound thyroxine by centrifugation and the concentration of free thyroxine determined from a standard curve prepared by use of known amounts of free thyroxine. The within-day coefficient of variation for two control samples was 8.8 and 6.8%; day-to-day precision for the same material was 12.5 and 13.5%. Lipemia, icterus, or hemoglobin had no adverse effect. Interlaboratory evaluation of the procedure revealed no significant difference in mean values for 20 speciments (p greater than 0.05).

Measurement of free thyroxine in human serum by a Sephadex binding method.

ABSTRACT A method of measuring free thyroxine (T4) is described which uses the property of Sephadex to bind T4. If 500 mg Sephadex G-25, 40 μI of serum in 4 ml physiological buffer, and 125I-T4 are mixed for 10 min at 37 C in a test tube, the unbound or free 125I-T4 in the aqueous phase is 12% of the 125I-T4 bound to Sephadex. This fraction depends specifically on the low affinity but high capacity of Sephadex for T4, and is unaffected by the amount of T4 or protein in the system although the amount of protein-bound 125I-T4 in the aqueous phase may change markedly. Since Sephadex-bound 125I-T4 is readily measured, the amount of free 125I-T4, and the fraction, free/total 125I-T4 in the dilute serum which forms the aqueous phase in the system are readily calculated. This “free T4 fraction” is related to the in vivo free T4 fraction only if the ratio, total T4: T4 binding protein is the same in this aqueous phase as it is in undiluted serum. These conditions are obtainable by experimental design or calculati...

Re-evaluation of thyroxine binding and free thyroxine in human serum by paper electrophoresis and equilibrium dialysis, and a new free thyroxine index.

ABSTRACT Thyroxine (T4) binding by 3 protein carriers (TBG, TBPA and albumin) was assessed in sera from various thyroid states using reverse flow paper electrophoresis, with which in normal serum 76% of endogenous T4 was bound to TBG, 15 % to TBPA and 9 % to albumin despite maximal TBG and TBPA capacities, consistent with previous reports. The percentage of endogenous T4 bound to TBG was increased to 87% in hypothyroidism and 88% in pregnancy, while it was decreased to 42 % in hyperthyroidism and 62 % in patients treated with anabolic steroids. Our results indicate that, in the above conditions, the role of TBG in T4 transport is even more important than has been previously recognized. On the basis of the newly estimated distribution of endogenous T4, the following relationship was established between free TBG capacity and triiodothyronine (T3) resin uptake (TRU): free TBG capacity (μg/100 ml) =5.02/ TRU(%/100)–4.98. Further, association constants of TBG and TBPA for T4, and free T4 concentrations were ca...

Radioiodinated impurities in commercial preparations of 131-I-thyroxine and their effect on the measurement of free thyroxine in human serum by equilibrium dialysis.

Chromatographic analysis of 9 lots of commercially prepared 131I-thyroxine indicated the presence of significant amounts of contaminating 131I-labeled 3,5,3′-triiodothyronine (average 3.7%) and 3,3′5′-triiodothyronine (average 5.4%). Since these iodothyronines are much less strongly bound to serum proteins than thyroxine, they would be expected to make a disproportionately high contribution to the radioactivity in the dialysate of dialysis systems used for measuring the free thyroxine concentration in serum. This expectation was experimentally confirmed. Direct chromatographic analysis of the dialysate demonstrated that a substantial proportion of the radioactivity was derived from the contaminating iodothyronines. Purification of 131I-thyroxine by paper chromatography, thinlayer chromatography, or preparative dialysis resulted in a 25% reduction in the dialyzable fraction as determined in the dialysis system employing diluted serum. Commercial preparations of 125I-labeled thyroxine were found to contain much smaller quantities of contaminating thyronines, and results of dialysis studies using this tracer approximated those obtained with purified 131I-thyroxine. Since the binding of the iodinated contaminating thyronines to serum proteins differs qualitatively from that of thyroxine, it is essential to use only the purest thyroxine preparation available in studies of thyroxine binding by serum proteins. The presence of these radioiodinated contaminants also may be the source of potential error in the interpretation of physiological studies which involve the use of commercial preparations of 131I-thyroxine.

Free thyroxine in human serum: simplified measurement with the aid of magnesium precipitation.

Free thyroxine in human serum: simplified measurement with the aid of magnesium precipitation.

DETERMINATION OF FREE THYROXINE IN HUMAN SERUM: A THEORETICAL AND EXPERIMENTAL ANALYSIS.

The effect of dilution of serum on the concentration of free thyroxine was evaluated in an effort to determine the relationship between values previously obtained in a standard dialysis system employing diluted serum (1:150) and the concentration of free thyroxine in undiluted system. A theoretical analysis of thyroxine-binding by serum proteins in terms of a multiple equilibria system suggested that the free thyroxine concentration should be relatively independent of the concentration of serum proteins in the range of dilution considered. In order to test this prediction, experimental techniques were devised to measure free thyroxine in dialysis systems containing higher concentrations of protein. For this purpose, tracer 131I-thyroxine and 125I-iodide were used simultaneously. The results of these studies were in general accord with the theoretical formulation. The concentration of free thyroxine was also measured in the ultrafiltrate of 7 sera. Values were in the same order of magnitude as those obtain...