Biomedical subjects
Publications and source records attributed to T Hachiya.
Because beta2-microglobulin is structurally similar to IgG, the reaction of beta2-microglobulin with Staphylococcal Protein A, which is known to react with the Fc region of IgG, was examined. 125I-beta2-microglobulin did not bind to Protein A. This may due to the difference in the amino acid sequence between beta2-microglobulin and the Fc region of IgG.
The effect of Fusaric acid (FA), a specific inhibitor of dopamine beta-hydroxylase, on humna TSH and thyroid hormone concentration (T4 and T3) was evaluated. Healthy subjects showed no significant changes in serum T3,T4 and TSH concentrations following the administration of FA calcium salt (FA-Ca) or placebo. Similarly, administration of FA-Ca for 4 weeks to hypertensive patients failed to produce significant changes in the serum T4 or T3 Resin Sponge Uptake values, and in the TSH and T3 responses to TRH. In contrast, FA-Ca produced a significant reduction on the high basal serum TSH level in patients with primary hypothyroidism. The mean nadir was 25% and ranged from 6 to 61%. As in the case of L-Dopa, the effect of FA-Ca on serum TSH is most clearly demonstrated in patients with primary hypothyroidism. Alterations in brain amines may directly or indirectly suppress pituitary TSH secretion. The possibility of changes in the peripheral distribution or turnover rate of TSH has not been excluded.
Three thyroid stimulators; LATS, TSH, and cholera toxin, have been tested by bioassay using congenitally athymic homozygous mice (nu/nu), nude mice and control littermates (nu/+) possessing normal thymic function. nu/+ Animals showed a response to LATS similarly to normal white mice of the ddN strain. The assay response to LATS in nu/nu animals was about half that in normal mice. TSH also showed a less stimulating action in nude mice. Cholera toxin made a long acting response similarly to LATS. The response to cholera toxin in nude mice was about half that of normal control mice. The thyroid of nude mice has a receptor for LATS-immunoglobulin, TSH or cholera toxin, which is similar to that of normal mice.
The immunological character of LATS was examined by affinity chromatography on Anti-IgG, Anti-Fab, Anti-Fc and Staphylococcal Protein A bound Sepharose. By affinity chromatography on Anti-IgG, Anti-Fab and Anti-Fc bound Sepharose, it is possible to separate LATS-immunoglobulin from LATS positive serum without loss of activity. Affinity chromatography on Protein A bound Sepharose is useful for obtaining further purified LATS-immunoglobulin. By this method, it is possible to separate IgG molecules of the subclasses IgG(1), IgG(2) and IgG(4) with high LATS activity. LATS activity was not found in the IgG(3) fraction. When IgG(1) fraction was purified from the fraction containing the 3 subclasses of IgG(1), IgG(2) and IgG(4), about 85% of total protein was found in IgG(1). However, specific activity per protein of LATS in IgG(1) fraction did not change remarkably. After papain hydrolysis the thyroid stimulating activity of LATS-immunoglobulin was located in Fab fraction of these 3 subclasses of IgG(1), IgG(2) and IgG(4), and especially in IgG(1). The Fab fraction presents a short acting type of thyroid stimulating activity. These data indicate that LATS activity is mainly distributed in the Fab fragment of IgG(1).
The reaction of LATS activity with Staphylococcal Protein A, a specific binding protein with the Fc part of human IgG(1), IgG(2) and Ig(4), was examined. When IgG(1), IgG(2) and Ig(4) subclasses were removed from LATS positive sera or LATS-IgG fractions by affinity chromatography on Protein A-Sepharose, LATS activity decreased. Almost all LATS activity was found in the fraction that reacted with Protein A. It is suggested that LATS has an expression of a very distinct immunoglobulin G structure, and that LATS activity is distributed mainly in the fraction containing IgG(1), Ig(2) and Ig(4) in LATS positive serum;
This study was undertaken to determine T3 content in red cells by radioimmunoassay. T3 in red blood cells was solubilized fairly from the stroma by hemolysis and red-cell T3 content could be determined directly by radioimmunoassay of the lysate. After hemolysing red cells with an equal volume of distilled water, 0.4 ml of the hemolyzate was used for the assay. The red-cell T3 content was expressed as ng/ml of red-cell volume. The normal T3 range in red cells was 0.20-0.45 ng/ml, and the Mean+/-SD was 0.32+/-0.10 ng/ml. The limit of detectability was 0.2 ng/ml. In hyperthyroid patients, the red-cell T3 content was more than 0.50 ng/ml with a Mean +/-SD of 1.35+/-0.65 ng/ml. In hypothyroid patients, red cells contained less than 0.25 ng/ml of T3, and there was an overlap from 0.20 to 0.25 ng/ml in the content of red-cell T3 in hypothyroid and euthyroid subjects. The patients with T3 toxicosis showed a high or normal level of red-cell T3. A positive correlation was noted between the red-cell T3 content and the serum T3 level (r=0.66). The correlation between the red-cell T3 content and the free T4 index (expressed as T7) was also positive (r=0.67). From these experiments, it is suggested that the red-cell T3 is low in comparison with the serum T3 levels, and depends on two factors; serum T4 and serum T3 levels.
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The determination method of c-AMP in urine by competitive protein binding assay using the Boehringer Mannheim Laboratory kit was investigated and the results obtained were as follows. 1) Since the angle of the inclination of the standard curve was large, the present method could be used for the determination of c-AMP in concentrations of from 0 to 20p moles. 2) The optimal condition of the binding reaction was for 100 minutes of reaction time at pH 4.0 and 4 degrees C. 3) The specificities of binding protein to the other nucleotides were 0 to adenosine, 0.4 to AMP, 0.3 to ADP, 0.4 to ATP and 0.6 to c-GMP respectively when the specificity to c-AMP was chosen as 100. 4) The optimal volume of cold phosphate buffer solution needed for washing to separate binding c-AMP by filtration method using a millipore filter was 5 approximately 15 ml. 5) The precision of the present method by double determination was 0 arrroximately +/- 11.1% with average of +/-5.6% in c.v. 6) The recovery rate of the added c-AMP by the present method was 78.6 approximately 105.6% with average of 90.1%. 7) Correlation between the determination values of c-AMP of the same samples with the present method and radioimmunosasay (Schwarzman Laboratory Kit) was satisfactorily high with 0.890 in coefficient of correlation, and the determination values by the present method were significantly higher (p less than 0.05) than those by radioimmunoassay. 8) C-AMP in urine was stable for at least one month when the urine was kept frozen. It is conclusively considered from the above results that this competitive protein binding assay is a method to determine c-AMP in urine with excellent accuracy and sensitivity and that this method is useful for clinical test.
A new method for the estimation of the maximal T4-binding capacity of TBG (max. TBG) in serum (dextran-coated charcoal-triosorb method) was devised. Thyroxine (T4) concentration in test sera were determined by the Tetrasorb method and then T3 resin sponge uptake (T3 RSU) was determined after extraction of 70% of the endogenous T4 with dextran-coated charcoal. The binding capacity of unsaturated TBG in treated sera was estimated from the reciprocal of T3 RSU. Max. TBG was calculated from the arithmetic sum of the unsaturated binding capacity of TBG and the serum T4 concentration multiplied by 0.3. The normal range for max. TBG was 20.1 +/- 2.6 (mean +/- SD) mug T4/100 ml (14 approximately 24) and it was 19.2 +/- 3.5 mug T4/100 ml in hyperthyroidism which was lower compared with hypothyroidism (21.1 +/- 2.6 mug T4/100 ml). Max. TBG in 8 hyperthyroid patients did not change significantly after treatment in half of the case, although in the other half it was slightly increased. Increased values of max. TBG were observed in pregnancy (32.9 +/- 4.4) and in a hyperthyroid state associated with pregnancy (31.4 +/- 4.7). Decreases were observed in TBG deficiency (less than 5.6 mug T4/100 ml). These procedures can be applied for the determination of both the unsaturated binding capacity of TBG and that of max. TBG.
Serum T3 level in various thyroid diseases was determined in unextracted serum with the Dainabot kit for T3 RIA. The serum T3 level in 33 normal subjects was 0.8-1.6 ng/ml. It was 5.7 +/- 3.5 ng/ml (mean +/- S.D.) in 36 hyperthyroid patients, and undetectable to 0.8 ng/ml in 21 hypothyroid patients. Generally the serum T4 and serum T3 decreased in parallel after radioiodine therapy for hyperthyroidism. However, in some cases the serum T3 level remained high in spite of normalized serum T4 after radioiodine therapy. This state indicated "T3-toxicosis", and hyperthyroidism was apt to recur. When thyroid function was observed for 2 years following radioiodine treatment, the ratio of serum T3 (T3 level before treatment/T3 level after treatment) decreased more significantly as compared with the ratio of serum T4 in euthyroid cases. Serum T3 provides a more sensitive index of thyroid function than serum T4 in euthyroid states after radioiodine or anti-thyroid drug therapy. The present data indicate that the serum T3 level and the T4/T3 ratio are valuable aids in the estimation of prognosis of hyperthyroid patients after various treatments.
A specific double antibody radioimmunoassay has been develop for the measurement of thyroglobulin in human serum. Human thyroglobulin was purified by combined DEAE-cellulose and affinity chromatography using Sepharose 4B-bound Concanavalin A. Sensitivity of test serum was 10 ng/ml. Thyroglobulin was not detectable in half of normal subjects, and half showed values between 10 and 180 ng/ml. In the patients with simple goiter and secondary hypothyroidism, serum thyroglobulin was usually in the normal range. In Hashimoto's thyroiditis, many sera having precipitating antibodies or high hemagglutination antibodies for thyroglobulin showed a high thyroglobulin concentration in serum probably due to a false positive reaction. In hyperthyroidism, an increased thyroglobulin level was observed in 64% of patients. However, there was no correlation between serum thyroglobulin and thyroxine levels in untreated hyperthyroidism. Serum thyroglobulin was increased significantly in some cases for several weeks after isotope therapy for the hyperthyroidism.
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