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PubMed2001

A new homogeneous enzyme immunoassay for thyroxine using glycogen phosphorylase b-thyroxine conjugates.

BACKGROUND: [corrected] Measurement of serum thyroxine (T(4)) concentration is important for diagnosis of thyroid gland diseases. We developed a practical homogeneous enzyme immunoassay for thyroxine analysis in unextracted sera. METHODS: A thyroxine derivative conjugated to a reactive sulfhydryl group of glycogen phosphorylase b (GPb). Conjugation caused inhibition of enzyme activity and the enzyme conjugate was re-activated upon the binding of a polyclonal anti-T(4) antibody. Antibody-activation was blocked by the presence of free T(4). RESULTS: Conjugation affected the allosteric character of the enzyme and the K(m) for the allosteric activator AMP was increased 28 times, while anti-T(4) antibody partially reversed this effect. The optimum concentration ratio of enzyme conjugate to anti-T(4) antibody was determined, and T(4) was measured with desired sensitivity and accuracy in the range between 10 and 240 microg/l. Furosemide was used to inhibit the interaction of thyroxine with serum T(4)-binding sites. Human serum T(4) values obtained by this method correlated well with those obtained by a radioimmunoassay (y=1.9+1.0x, r=0.97, N=72). CONCLUSIONS: Chemical modification of glycogen phosphorylase b with a T(4) derivative led to the development of a simple homogenous enzyme immunoassay for T(4) analysis with the desired sensitivity and accuracy.

Allosteric Regulation↗
PubMed1981

A comparison of free thyroxine concentration and the free thyroxine index as diagnostic tests of thyroid function.

Serum free thyroxine concentrations, measured by the Immo Phase kit, and free thyroxine index values were compared in 200 subjects classified according to age, sex, and clinical diagnosis. The free thyroxine concentration was as good as the free thyroxine index in hyperthyroid, hypothyroid, elderly, and acutely ill patients and a better diagnostic index of thyroid status in pregnancy and in oral contraception.

Acute Disease↗
PubMed1979

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.

Humans↗
PubMed1993

Establishment of reference ranges for thyrotropin, triiodothyronine, thyroxine and free thyroxine in neonates, infants, children and adolescents.

Thyrotropin, triiodothyronine, thyroxine and free thyroxine were determined in the sera of 714 euthyreotic neonates, infants, children and adolescents (age range 5 days to 18 years), using the IMx from Abbott Laboratories. Thyrotropin, triiodothyronine and free thyroxine were determined with microparticle enzyme immunoassays (MEIA). Thyroxine was determined with a fluorescence polarization immunoassay (FPIA). The proband collective was divided into 9 age groups, and each age group into males and females. In accordance with the recommendations of the International Federation of Clinical Chemistry, the 95% scatter range was taken as the reference range. In the different age groups, the ranges of some hormones showed significant differences between males and females. When no significant difference existed between the sexes, the results for males and females were evaluated statistically as a single group. Only a few reference groups showed normal Gaussian distributions. Therefore, in addition to the 50th percentile, the 2.5th and 97.5th percentiles were also calculated for all reference groups. Minimal and maximal values were also determined. The U-test of Mann & Whitney was used to test for significant differences between individual reference groups, and groups showing no significant differences were combined. The corresponding references ranges were then calculated.

Adolescent↗
PubMed1982

Effect of anti-thyroxin autoantibodies on radioimmunoassay of free thyroxin in serum.

Serum thyroxin was nearly or completely undetectable by radioimmunoassay in an elderly patient with Graves' disease being treated with methimazole. Abnormal binding of thyroxin to antibodies of the IgG variety was shown, the association constant of the complex being 8.0 x 10(8) L/mol and the binding capacity 6.3 nmol/g of IgG. The effect of the antibody on results of radioimmunoassay of free thyroxin was studied with three commercial kits, two of which (Clinical Assays and Damon Diagnostics) gave essentially the same values as did equilibrium dialysis. The third (Amersham International) gave falsely high results because the 125I-labeled thyroxin derivative used in the kit was bound by the autoantibody.

Autoantibodies↗
PubMed1976

TRH induced thyroxine release from thyroid: its prevention by thyroxine influenced by actinomycin D and cycloheximide.

Male rats of a final weight 350-400 g were fed low iodine diet for 4 weeks and injected radioiodide for 6 days prior to the experiment. They were anesthetized with pentobarbiturate and external arteriovenous shunt was made with the aid of thin polyethylene tubing to collect frequent blood samples while maintaining isovolemia as previously described. The injection of 20 mug synthetic TRH into carotic artery in rostral direction resulted in an increase of plasma thyroxine radioactivity showing a maximum at 120 min after the injection. This increase was prevented with the aid of i.v. injection 20 mug L-thyroxine at 20-180 min before TRH administration. Moreover, the preventive effect of thyroxine was completely blocked by the i.v. injection of actinomycin D (0.8 mg/kg) 60 min before thyroxine, while the administration of cycloheximide (4 mg/kg), at the same time, was without effect. The mechanism of this phenomenon remains to be further elucidated.

Animals↗
PubMed1985

Further evidence for the presence of thyroxine binding globulin-like protein in human breast adipose tissue. Deiodination of thyroxine and triiodothyronine by the microsomal fraction.

The object of the study was to obtain information on how adipose tissue of normal subjects processes thyroid hormones. L-125I-thyroxine(125I-T4) is bound by the cytosol fraction of normal human female breast adipose tissue with high affinity. Computer analysis of the binding data revealed the presence of two saturable systems with Kd values of 3.9 and 29.1 nM and binding capacities of 1.7 and 8.7 pmol/mg of cytosol protein, respectively; a third binding system was non-saturable. The binding of the iodothyronines to the cytosol fraction indicated that L-triiodothyronine (T3) possessed two-fold higher affinity as compared to L-thyroxine (T4) whereas other iodothyronines had relative affinities of less than 3%. Binding of 125I-T4 was optimal at pH 7.0 and was sensitive to the action of pronase and neuraminidase. Affinity chromatography of the cytosol fraction using T3-epoxy-sepharose 6B and con A-sepharose 4B, yielded a 125I-T4 binding component that was purified 150-fold. Isoelectric focusing of the purified fraction yielded six major brands of protein which had pI values comparable to those of human serum thyroxine-binding-globulin (TBG); however, the pattern of separation was different. Incubation of the fraction with 125I-T4 followed by isoelectric focusing and autoradiography revealed that the protein bands bound radioactivity. The microsomal fraction of the adipose tissue deiodinated 125I-T4 to L-125I-triiodothyronine (125I-T3) to an extent of 0-0.8 fmol/(mg of protein X min) which corresponded to about 0.2%; T4 was not deiodinated to L-3,3',5'-triiodothyronine (r-T3).(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1989

Binding activities of thyroxine binding globulin versus thyroxine binding prealbumin in rat sera: differential modulation by thyroid hormone ligands, oleic acid and pharmacological drugs.

We use gel equilibration and electrophoretic techniques to compare the binding properties of thyroxine binding globulin and thyroxine binding prealbumin in rat sera. The evidence indicates that TBG bears the serum lowest capacity highest affinity sites for thyroxine (T4) and triiodothyronine (T3) (Ka1 greater than or equal to 10(9) M-1) as well as weaker saturable T3 sites (Ka2 approximately 10(8) M-1). TBPA bears for T4 only Ka2 approximately 10(8) M-1 sites and for T3 only Ka approximately 10(6) M-1 sites. Consistent with these parameters are the specific responses of TBG and TBPA binding activities to varying serum concentrations of T4, T3, oleic acid, the drugs diphenylhydantoin or salicylate. The primary attack of these compounds is aimed at TBG. Small T4, oleate or DPH doses chase the TBG-bound T4 to TBPA, high doses of T4 or oleate but not of DPH inhibiting the T4 binding to both proteins. In the T3-serum interactions, all tested compounds displace the TBG-bound hormone without chasing it to TBPA. The high reactivity of TBG sites designates the protein as crucially involved in modulating the free vs bound serum levels of T4 and T3 against physiological or pathological variations of binding competitors.

Animals↗
PubMed1989

Thyroxine-binding globulin and thyroxine-binding prealbumin in hypothyroid and hyperthyroid developing rats.

We present evidence based on equilibrium and non-equilibrium binding studies, as well as on immunological techniques, that of the two rat specific thyroid-hormone-binding proteins, i.e., thyroxine-binding globulin (TBG) and thyroxine-binding prealbumin (TBPA), TBG but not TBPA is regulated by the thyroid hormones (TH). Hypothyroidism, induced from the day of birth by daily treatment with propylthiouracil (PTU-rats), leads to dramatic and sustained increases of the TH-binding abilities of the sera measured at equilibrium, whereas hyperthyroidism, induced by treatment with thyroxine (T4-rats), leads to the decrease of these abilities. Polyacrylamide gel electrophoresis and isoelectrofocalisation of radioiodinated T4-labelled sera, together with immunoassay of TBPA, demonstrate that both effects are due to TBG, the levels of which rise in PTU-rats and decline in T4-rats, while TBPA levels do not respond to either depletion or excess of the thyroid hormones. TBG rather than TBPA appears as the key thyroid-hormone-binding protein of the rat, inasmuch as it alone expresses a regulatory function of the thyroid hormones at protein synthesis level.

Aging↗
PubMed1974

Comparison between determination of free thyroxine index and effective thyroxine ratio in human serum.

Four thyroid function indices were determined in the sera of 181 patients: total thyroxine (T-4), the triiodothyronine resin uptake (T-3 B.C.-index), the free thyroxine (T-4) index, and the effective thyroxine ratio (E.T.R.).A statistically significant correlation between the free T-4 index and E.T.R. could be established though this correlation seemed to be worse than could be expected from the literature. Two sets of correlation coefficients were shown: one obtained with the ordinarily used Pearson or product-moment correlation and one with the Spearman rank correlation. Because the assumptions and requirements for the proper use of the product-moment correlation statistics, such as a normal distribution of both variables, are seldom met, expecially not if a selected number of controls and patients is used for the correlation, it is advisable to use some kind of rank correlation statistics instead.The advantages and disadvantages of the E.T.R. and free T-4 index determination are described.

Blood Proteins↗
PubMed1972

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.

Aspirin↗
PubMed1982

Peripheral serum thyroxine, triiodothyronine and reverse triiodothyronine kinetics in the low thyroxine state of acute nonthyroidal illnesses. A noncompartmental analysis.

The low thyroxine (T(4)) state of acute critical nonthyroidal illnesses is characterized by marked decreases in serum total T(4) and triiodothyronine (T(3)) with elevated reverse T(3) (rT(3)) values. To better define the mechanisms responsible for these alterations, serum kinetic disappearance studies of labeled T(4), T(3), or rT(3) were determined in 16 patients with the low T(4) state and compared with 27 euthyroid controls and a single subject with near absence of thyroxine-binding globulin. Marked increases in the serum free fractions of T(4) (0.070+/-0.007%, normal [nl] 0.0315+/-0.0014, P < 0.001), T(3) (0.696+/-0.065%, nl 0.310+/-0.034, P < 0.001), and rT(3) (0.404+/-0.051%, nl 0.133+/-0.007, P < 0.001) by equilibrium dialysis were observed indicating impaired serum binding. Noncompartmental analysis of the kinetic data revealed an increased metabolic clearance rate (MCR) of T(4) (1.69+/-0.22 liter/d per m(2), nl 0.73+/-0.05, P < 0.001) and fractional catabolic rate (FCR) (32.8+/-2.6%, nl 12.0+/-0.8, P < 0.001), analogous to the euthyroid subject with low thyroxine-binding globulin. However, the reduced rate of T(4) exit from the serum (Kii) (15.2+/-4.6 d(-1), nl 28.4+/-3.9, P < 0.001) indicated an impairment of extravascular T(4) binding that exceeded the serum binding defect. This defect did not apparently reduce the availability of T(4) to sites of disposal as reflected by the increased fractional disposal rate of T(4) (0.101+/-0.018 d(-1), nl 0.021+/-0.003, P < 0.001). The decreased serum T(3) binding was associated with the expected increases in MCR (18.80+/-2.22 liter/d per m(2), nl 13.74+/-1.30, P < 0.05) and total volume of distribution (26.55+/-4.80 liter/m(2), nl 13.10+/-2.54, P < 0.01). However, the unaltered Kii suggested an extravascular binding impairment comparable to that found in serum. The decreased T(3) production rate (6.34+/-0.53 mug/d per m(2), nl 23.47+/-2.12, P < 0.005) appeared to result from reduced peripheral T(4) to T(3) conversion because of decreased 5'-deiodination rather than from a decreased T(4) availability. This view was supported by the normality of the rT(3) production rate. The normal Kii values for rT(3) indicated a comparable defect in serum and extravascular rT(3) binding. The reduced MCR (25.05+/-6.03 liter/d per m(2), nl 59.96+/-8.56, P < 0.005) and FCR (191.0+/-41.19%, nl 628.0+/-199.0, P < 0.02) for rT(3) are compatible with an impairment of the rT(3) deiodination rate. These alterations in thyroid hormones indices and kinetic parameters for T(4), T(3), and rT(3) in the low T(4) state of acute nonthyroidal illnesses can be accounted for by: (a) decreased binding of T(4), T(3), and rT(3) to vascular and extravascular sites with a proportionately greater impairment of extravascular T(4) binding, and (b) impaired 5'-deiodination activity affecting both T(4) and rT(3) metabolism.

Acute Disease↗
PubMed1981

Ratio of thyroxine to thyroxine-binding globulin. An assessment of the validity of a single reference range.

The thyroxine:thyroxine-binding globulin ratio in serum (T4:TBG) has been proposed as a measure of thyroid status unaffected by altered binding protein levels. Here 320 apparently euthyroid patients are used to derive euthyroid ranges for serum thyroxine concentrations at specific TBG levels. These ranges are compared with those predicted by a T4:TBG reference range derived from the same patient data. The comparison suggests that the ratio would give false positives for hyperthyroidism at low TBG levels and false negatives at high TBG levels. To overcome this the use of graphical reporting of results or the relation of patient results to empirical reference ranges appropriate to the TBG level is suggested.

Alpha-Globulins↗
PubMed1983

Serum free thyroxine concentrations in subjects with high circulating levels of thyroxine-binding globulin.

Serum free thyroxine (FT4) and thyroid hormone-binding protein concentrations were measured in pregnant women, stilboestrol-treated patients, and subjects with congenital thyroxine-binding-globulin (TBG) excess. In pregnant and stilboestrol-treated patients matched for TBG concentrations, serum FT4 concentrations measured by the Amerlex technique and by calculation, were reduced, as also was the total thyroxine (TT4)/TBG ratio. In congenital TBG excess Amerlex and calculated FT4 concentrations were normal whereas the TT4/TBG ratio was low. In contrast to the Amerlex method, FT4 concentrations by the Immophase procedure were normal in pregnant and stilboestrol-treated individuals and high in congenital TBG excess. We conclude that (i) reduced FT4 concentrations in pregnancy are related to an oestrogen effect rather than to some other pregnancy-associated factor, (ii) the TT4/TBG ratio gives a misleading indication of FT4 concentration in congenital TBG excess, and (iii) that when TBG concentrations are raised Immophase gives higher FT4 values than Amerlex or calculation when each is compared to FT4 values in their normal respective control group.

Adult↗
PubMed1975

Serum thyroxine and thyroxine-binding proteins in chronic renal failure without nephrosis.

Total serum thyroxine (T4), free thyroxine index (FTI), thyroxine binding globulin (TBG) binding capacity, serum albumin, alpha-globulins and urinary protein excretion were measured in 50 patients with chronic renal failure, but without nephrotic syndrome. 25 patients were undergoing chronic hemodialysis. T4 was within the normal range in most patients. There was a tendency to lower T4 values as compared to an age and sex-matched control group, but this did not reach statistical significance. TBG was normal in most patients. 4 patients showed elevated TBG concomitant with elevation of other alpha-globulins. Serum albumin was significantly decreased. No correlation existed between daily protein excretion and TBG or alpha-globulins, but the correlation between serum albumin and proteinuria was highly significant. T4 and proteinuria correlated with borderline significance. A highly significant correlation between T4 and TBG-albumin values was found. No correlation existed between FTI and TBG-albumin levels. The data suggest that T4 and TBG are normal in most patients with renal failure, even in the presence of significant proteinuria. Low T4 values, when found in renal insufficiency, may be secondary to low serum albumin and possibly prealbumin.

Adult↗
PubMed1976

Increased free thyroxine in a euthyroid patient with thyroxine-binding globulin deficiency.

An unusual finding of elevated free thyroxine (FT4) concentration coexistent with familial low thyroxine-binding globulin (TBG) is described in a euthyroid patient. Clinical and other laboratory tests were normal and consistent with a clinical diagnosis of euthyroidism. Low TBG (TBG capacity 3-10 mug/100 ml) was confirmed by various electrophoretic procedures and by TBG immunoassay (0.9 mg/100 ml). Serum T4 concentration was low (2.0-5.5 mug/100 ml). Triiodothyronine (T3) resin uptake was increased (88-93%) and the free thyroxine index was normal. Twenty-four-hour thyroidal 131I uptake was normal and varied between 13-20%. Serum thyroid-stimulating hormone (less than 2 muunits) and T3 concentration (80 ng/100 ml) were also normal. Long-acting thyroid stimulator (LATS) was not present. Free T4 concentration measured by dialysis procedure was markedly elevated (9.2-12.1 ng/100 ml). Pulse T3 and T4 tracer kinetic studies by the single compartmental method revealed accelerated blood disappearance of T4 (t 1/2 = 3.7 days) but normal disappearance of T3 (t 1/2 = 2.1 days). Extrathyroidal hormonal iodine pool (569 mug) was in the range of normals. T4 degradation rate (144 mug/70 kg/day) was moderately elevated over the euthyroid mean values, but was still close to or within the upper normal range. T3 degradation was normal (34 mug/day). Failure to develop hyperthyroidism in the presence of elevated free T4 levels, but with normal T3 concentration and dynamics, may suggest that T3, not T4, plays a major role in regulating metabolic activity; alternatively, euthyroidism in this low-TBG patient despite elevated free T4 concentration may simply be explained on the basis of a daily T4 disposal rate which was not clearly abnormal.

Humans↗
PubMed1977

Changes in serum levels of thyroxine and thyroxine-binding proteins (TBG, TBPA, ALBUMIN) induced by venous stasis.

Serum concentrations of thyroxine (T4), thyroxine-binding globulin (TBG), thyroxine-binding pre-albumin (TBPA) and albumin were determined in 21 healthy, young subjects before and after a brief venous stasis in two experiments: 1) 3 min stasis induced by a sphygmomanometer with constant pressure 20 mmHg above the diastolic blood pressure and 2) 2 min stasis induced by an arm tourniquet of rubber. In both experiments the serum T4 level was significantly rised (mean 9%) after venous compression. Increases of the same magnitude were observed for serum TBG, serum TBPA and serum albumin. The serum concentrations of the free constituents--sodium and creatinine--remained unchanged, whereas the haemoglobin concentration increased (mean 8%). This haemoconcentrating effect of venous stasis seemed to be more pronounced in females than in males. Our data emphasize the need for protein correction procedures when total serum T4 is measured.

Adult↗
PubMed1987

Serum concentrations of thyrotropin, thyroxine, triiodothyronine and thyroxine binding globulin in female endurance runners and joggers.

The effects of endurance training and season on the function of the anterior pituitary-thyroid axis were studied in 18 female runners and their 12 controls, and in 13 joggers and their 11 controls in Northern Finland, with a large seasonal difference in environmental factors. The serum concentrations of thyrotropin (TSH), thyroxine (T4), free thyroxine (fT4), triiodothyronine (T3), thyroxine binding globulin (TBG) and oestradiol (E2) were measured during one menstrual cycle in the light training season (autumn) and in the hard training season (spring). The responses of TSH to intravenous TRH stimulation were also measured in the luteal phase of the cycle during the hard training season. Endurance running did not affect the basal or TRH-stimulated serum TSH concentrations, while those of T4 and fT4 in runners were lowered in both seasons and that of T3 in the light training season in relation to control subjects. The serum concentrations of TBG were also significantly lower in runners than their controls in the luteal phase in both seasons. The effect of jogging on thyroid hormones was less pronounced. Serum concentrations of TSH, T4, fT4, T3 and TBG were generally slightly higher in spring than in autumn. Strenuous endurance training seems to have minor changes on the function of the thyroid gland. Depressed T4 levels in runners may rather be due to lowered TBG levels than due to direct effect of training. In spring the function of anterior pituitary-thyroid axis is more active than in autumn.

Adult↗