Kiyoshi Ichihara

ASYMPTOMATIC HYPERPROLACTINAEMIA AND PROLACTINOMA IN THE GENERAL POPULATION—MASS SCREENING BY PAIRED ASSAYS OF SERUM PROLACTIN

A total of 10 550 ‘normal’ adults (8450 men and 2100 women) in the general population were screened by a paired assay method for serum PRL and 40 subjects with hyperprolactinaemia (>75 μg/1) were detected. Of these, five patients (three men and two women) with pituitary prolactinoma, one man with empty sella syndrome, 10 cases of ‘big’ prolactinaemia, seven pregnant women and 13 subjects with drug‐induced hyperprolactinaemia were found. The patients with prolactinoma had few if any complaints (asymptomatic prolactinoma).

Longitudinal study or serum thyroid hormones, chorionic gonadotrophin and thyrotrophin during and after normal pregnancy.

Measurements of serum levels of thyroxine (T4), free T4, 3,5,3′‐triiodothyronine (T3), free T3, 3,3′,5′‐triiodothyronine (reverse T3, rT3), thyroxine‐binding globulin capacity (TBGcap), chorionic gonadotrophin (hCG) and thyrotrophin (TSH) were carried out prospectively in eight women with uncomplicated pregnancies, in order to examine interrelationships between the thyroid gland and thyroid stimulating hormones during pregnancy. During pregnancy the levels of T4, free T4, T3, rT3 and TBGcap were significantly elevated, and TSH was decreased. It was noted that the elevation of T4 was maintained from the 8th to the 27th week of gestation while the level of TBGcap progressively increased. The levels of free T4 and rT3 in the first and third trimesters were significantly higher than those of age‐matched, non‐pregnant women. The levels of hCG showed a biphasic variation, with a peak in the 8th to 15th weeks, followed by a decline in the second trimester and a small, secondary elevation in the 32nd to 39th weeks. This later elevation was positively correlated with changes in free T4 and free T3 levels. The increase of serum T4 accompanied by an increase of free T4 in the first trimester appeared due to augmented secretion of T4, rather than being secondary to the elevated levels of TBGcap.

Immunological and biological characteristics of recombinant human thyrotropin

Analyses of epitopes and biological activity were made on two preparations of recombinant human thyrotropin produced in Chinese hamster ovary cells. Seven monoclonal antibodies recognizing four epitopes on alpha subunit of hTSH (hTSH alpha) and three on beta subunit (hTSH beta) were used for the analysis. Binding activities of the two rhTSH with each antibody were almost identical with that of a pituitary-derived reference hTSH, except at one epitope on hTSH alpha. Their immunoreactivity measured by four commercial immunoassay kits and their bioactivity by thyrotropin dependent FRTL-5 cell system, however, agreed closely with those of the reference hTSH. From these results and its constant availability, rhTSH will be a good candidate for a future standard material in assays for hTSH.

Evaluation of TSH receptor antibody by 'natural in vivo human assay' in neonates born to mothers with Graves' disease.

Neonatal thyrotoxicosis induced by transferred TSH receptor antibody (TRAb) is the ideal human in‐vivo experimental system for the evaluation of TRAb. The clinical significance of circulating TRAb in Graves’ disease was evaluated by this ‘natural in‐vivo human assay’. TRAb activity in vitro was measured by radioreceptor assay (thyrotrophin‐binding inhibitor immunoglobulin, TBII) and sensitive cAMP accumulation assay using FRTL‐5 cells (thyroid‐stimulating antibody, TSAb). Further, the binding‐stimulation index (B‐S index) was newly introduced, which was the most useful indicator for prediction of neonatal thyrotoxicosis, calculated as the product of TBII and TSAb (Tamaki et al., 1988a). Maternal serum TRAb indices showed highly significant correlations with the serum free T4 index (FT4I) and free T3 index (FT3I) in neonates (5–10 days after birth) born to 20 mothers with Graves’ disease who had positive TBII and/or TSAb (FT4I: r= 0.825 for TBII, r= 0.908 for TSAb, r= 0.944 for the B‐S index, P > 0.001; FT3I: r = 0.622 for TBII, P > 0.01, r= 0.812 for TSAb, r= 0.791 for the B‐S index, P > 0.001; n= 20). In contrast, in 57 untreated adult patients with hyperthyroid Graves’ disease, the FT4I and FT3I levels were not correlated with any of the TRAb indices. The linear regression relationship between the B‐S index and FT4I found in neonates was applied to values in adult patients with Graves’ disease, and the patients were divided into three groups on the basis of the 95% confidence limit: high, normal, and low responders of thyroid hormone (FT4I) secretion to the B‐S index. FT4I and the ratio of FT4I to the B‐S index were highest and the TRAb indices were lowest in the high responders, while FT4I and the FT4I/B‐S index ratio were lowest and the TRAb indices were highest in the low responders. The FT4I/B‐S index ratio was inversely correlated with the titres of antithyroid microsomal antibody in all the adult patients with untreated Graves’ disease (r=−0.288, P >0.05). The results suggest that in‐vitro assays using animal thyroid cells and cAMP as an index of response are suitable for detecting circulating thyroid stimulating activity in vivo. Secretion of thyroid hormones in Graves’ disease may be regulated not only by circulating thyroid‐stimulating antibodies but also by intrathyroidal stimulatory factors or by inhibitory or destructive factors.

Seasonality of birth in sporadic cretinism

The seasonal distribution of birth dates of 31 patients with sporadic cretinism due to thyroid dysgenesis was analyzed in Osaka area for 14 years. The incidence was statistically high in the summer months. A hypothesis that some environmental factors such as viral infection may cause the disease is proposed.

The stability of immunological and biological activity of human thyrotropin in buffer: its temperature-dependent dissociation into subunits during freezing.

The stability of thyroid-stimulating hormone (TSH) in buffer under various storage conditions was studied with regard to its immunoreactivity measured by an immunoradiometric assay and its bioactivity by a sensitive FRTL-5 cell bioassay. The immunoreactivity was well retained at 4 degrees C or 24 degrees C throughout the study period of 90 days. At -20 degrees C, however, it decreased proportionately with the storage time. The mean reduction was 42.1% at 90 days compared with that when stored at -80 degrees C. The bioactivity showed a similar course of change with its reduction of 44.3% at -20 degrees C in 90 days. The loss of both activities was attributed to the dissociation of human (h) TSH molecule into its subunits. The concentration of the alpha subunit of hTSH in those samples stored at -20 degrees C gradually increased from the initial undetectable level to that almost equivalent on a molar basis to the loss of immunoreactivity. The enrichment of albumin in the buffer to a level of more than 1.0% was effective in preventing the occurrence of such a phenomenon. These data indicate that hTSH, when frozen at -20 degrees C in buffer is gradually dissociated into its subunits, despite its outstanding stability for 90 days at both 4 degrees C and 24 degrees C. However, no apparent inconsistency between immunological and biological activities, was observed at any temperature between -20 degrees C and 24 degrees C.

An improved processing of radioimmunoassay data by means of a desk-top calculator (1) comparison of regression procedures applied to selected kinds of radioimmunoassay

By means of a programmable desk-top calculator, goodness-of-fit of 3 regression models, four-parameter logistic, quadratic logit-log and linear logit-log models, were evaluated by analysis of variance (F test) for data of 6 kinds of radioimmunoassays (RIA); thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), insulin (IRI), cortisol, triiodothyronine (T3). Scatchard plot analyses were made with the representative data of these RIAs in order to find the best choice of regression model in relation to the characteristics of antigen-antibody reaction. The analysis of goodness-of-fit of the regression models by means of an F test disclosed the relation between the choice of regression models and the kinds of RIA, which could be grouped into 3 types: (1) almost identical degree of fit with any of 3 regression models (FSH and T3), (2) more or less equal degree of satisfactory fit with the logistic and quadratic logit-log models (TSH and cortisol), (3) best degree of fit with the aquadratic logit-log model among 3 (LH and IRI). The analysis of data with Scatchard plot discriminated 3 general types of curves; (1) linear (FSH) and T3), (2) linear with tail (TSH and cortisol) and (3) hyperbola (LH and IRI). From these findings, the following tentative conclusions were reached: RIA with linear pattern on Scatchard plot can be satisfactorily regressed with either of 3 models, RIA with linear with tail pattern regressed with either the logistic or quadratic logit-log model, and RIA with hyperbolic pattern regressed best with the quadratic logit-log model.

Reduced Serum Carnosinase Activity in Hypothyroidism

Carnosinase hydrolyses carnosine in muscle, and its deficiency is associated with extensive neuromuscular abnormalities. We measured serum carnosinase activity in patients with thyroid dysfunction which often involves neuromuscular systems. In hyperthyroidism, the carnosinase activity was not significantly different from that in normal subjects. In hypothyroidism, however, it was significantly lower than that in normal subjects. The activity examined in five patients with hypothyroidism returned to normal after replacement therapy. In hypothyroidism, the carnosinase activity showed significant correlation with concentration of serum thyroxine and negative correlation with serum creatine kinase activity. This finding may be of practical importance in the differential diagnosis of disorders causing carnosinase deficiency.

Fractionation of polyclonal antibody by isoelectric focusing and chromatofocusing: Separation of high-affinity rabbit clonotype anti-thyroxine antibody

Immunoglobulin G fractions prepared from conventional rabbit anti-thyroxine (T4) antisera were fractionated by agarose gel isoelectric focusing (IEF) in the range of pH 3 to 10, and by chromatofocusing using a Fast Protein Liquid Chromatography (FPLC) system. The clonotype antibodies were recovered from the fractions and subjected to Scatchard plot analysis. The highest affinity constants of the initial antibody (shown in parentheses) and those of the antibodies recovered were IEF, 1.8 X 10(9) to 8.3 X 10(9) M-1 (2.2 X 10(9) M-1); FPLC, 2.4 X 10(9) to 6.0 X 10(9) M-1 (2.5 X 10(9) M-1). A sensitive radioimmunoassay of T4 was achieved with the isolated high-affinity anti-T4 antibody. The minimum detectable concentration of T4 was 6.3 X 10(-15) to 1.5 X 10(-14) mol/tube, which was three to five times lower than detectable with the initial antibodies.

Fractionation of polyclonal antibody by isoelectric focusing—Differences in cross-reactivity and affinity of rabbit clonotype anti-human thyrotropin antibody

Immunoglobulin G (IgG) fractions prepared from three different batches of rabbit antihuman thyrotropin (hTSH) antisera were fractionated by agarose isoelectric focusing (IEF) in the pH ranges 3 to 10 and 5 to 8. Staining of protein in agarose gel after IEF showed that polyclonal IgG separated into more than 20 protein bands with isoelectric points (pIs) ranging from 6 to 9. The clonotype antibodies to hTSH were recovered from the fractions and subjected to radioimmunoassay for determination of the binding-affinity for hTSH and the cross-reactivity with human chorionic gonadotropin (hCG). The affinity constants of the antibodies recovered ranged from 6.4 X 10(9) M-1 to 3.1 X 10(10) M-1, and the cross-reactivities of the clonotype antibodies differed greatly. A good correlation was observed between the pIs of antibody molecules and their cross-reactivities: antibodies with higher pIs bound hCG more strongly than those with lower pIs. The correlation coefficients between the pIs and cross-reactivities were 0.83, 0.84, and 0.87 in three batches of antibody.