Bidirectional light-induced conductance switching in molecular wires containing a dimethyldihydropyrene unit.

Photochromic coordination polymers, based on zinc(ii) bis-terpyridine-appended dimethyldihydropyrene building blocks, have been synthesized following stepwise synthesis on a surface yielding photo-switchable molecular junctions. Under irradiation, reversible structural changes occur by the isomerization of the photosensitive units, thus inducing conductance switching of the molecular junctions with a good reproducibility.

Electrochemical control of the switching process of photochromic dimethyldihydropyrene derivatives.

The electrochemical control of the isomerization process of a photo-switchable dimethyldihydropyrene (DHP) derivative has been investigated. Spectro-electrochemical experiments clearly evidence that the substitution of the DHP core by electron-withdrawing entities can be used to control the opening/closing process.

Self-assembled dimethyldihydropyrene-pyridyl substituted ligands with zinc(ii) meso-tetraphenylporphyrin via axial coordination.

A series of dimethyldihydropyrene (DHP)-pyridyl photochromic derivatives has been synthesized and its photochemical behaviour characterized by spectroscopic and electrochemical methods. The corresponding noncovalently-linked electron donor-acceptor complexes have been isolated. They combine the DHP-pyridyl ligand as a donor and the zinc(ii) tetraphenylporphyrin as acceptor. Such association allowed to explore the efficiency of dative bonds to monitor the interactions between the two units.

Dicopper(II) complexes of H-BPMP-type ligands: pH-induced changes of redox, spectroscopic ((19)F NMR studies of fluorinated complexes), structural properties, and catecholase activities.

Substitution of the methyl group from the H-BPMP (HL(CH)3) ligand (2,6-bis[(bis(2-pyridylmethyl)amino)methyl]-4-methylphenol) by electron withdrawing (F or CF(3)) or electron donating (OCH(3)) groups afforded a series of dinucleating ligand (HL(OCH)3, HL(F), HL(CF)3), allowing one to understand the changes in the properties of the corresponding dicopper complexes. Dinuclear Cu(II) complexes have been synthesized and characterized by spectroscopic (UV-vis, EPR, (1)H NMR) as well as electrochemical techniques and, in some cases, by single-crystal X-ray diffraction: [Cu(2)(L(OCH)3)(muOH)][(ClO(4))(2)].C(4)H(8)O, [Cu(2)(L(F))(muOH)][(ClO(4))(2)], [Cu(2)(L(F))(H(2)O)(2)][(ClO(4))(3)].C(3)D(6)O, and [Cu(2)(L(CF)3)(H(2)O)(2)][(ClO(4))(3)].4H(2)O. Significant differences are observed for the Cu-Cu distance in the two mu-hydroxo complexes (2.980 A (R = OCH(3)) and 2.967 A (R = F)) compared to the two bis aqua complexes (4.084 A (R = F) and 4.222 A (R = CF(3))). The mu-hydroxo and bis aqua complexes are reversibly interconverted upon acid/base titration. In basic medium, new species are reversibly formed and identified as the bis hydroxo complexes except for the complex from HL(CF)3 which is irreversibly transformed near pH = 10. pH-driven interconversions have been studied by UV-vis, EPR, and (1)H NMR, and the corresponding pK are determinated. In addition, with the fluorinated complexes, the changes in the coordination sphere around the copper centers and in their redox states are evidenced by the fluorine chemical shift changes ((19)F NMR). For all the complexes described here, investigations of the catechol oxidase activities (oxidation of 3,5-di-tert-butylcatechol to the corresponding quinone) are of interest in modeling the catecholase enzyme active site and in understanding aspects of structure/reactivity. These studies show the pH-dependence for the catalytic abilities of the complexes, related with changes in the coordination sphere of the metal centers: only the mu-hydroxo complexes from HL(CH)3, HL(F), and HL(OCH)3 exhibit a catecholase activity. Modification on R-substituent induces a drastic effect on the catecholase activity: the presence of an electron donating group on the ligand increases this activity; the reverse effect is observed with an electron withdrawing group.

pH-controlled change of the metal coordination in a dicopper(II) complex of the ligand H-BPMP: crystal structures, magnetic properties, and catecholase activity.

The dinucleating ligand 2,6-bis[(bis(2-pyridylmethyl)amino)methyl]-4-methylphenol (H-BPMP) has been used to synthesize the three dinuclear Cu(II) complexes [Cu2(BPMP)(OH)][ClO4](2).0.5C4H8O (1), [Cu2(BPMP)(H2O)2](ClO4)(3).4H2O (2), and [Cu2(H-BPMP)][(ClO4)4].2CH3CN (3). X-ray diffraction studies reveal that 1 is a mu-hydroxo, mu-phenoxo complex, 2 a diaqua, mu-phenoxo complex, and 3 a binuclear complex with Cu-Cu distances of 2.96, 4.32, and 6.92 A, respectively. Magnetization measurements reveal that 1 is moderately antiferromagnetically coupled while 2 and 3 are essentially uncoupled. The electronic spectra in acetonitrile or in water solutions give results in accordance with the solid-state structures. 1 is EPR-silent, in agreement with the antiferromagnetic coupling between the two copper atoms. The X-band spectrum of powdered 2 is consistent with a tetragonally elongated square pyramid geometry around the Cu(II) ions, in accordance with the solid-state structure, while the spectrum in frozen solution suggests a change in the coordination geometry. The EPR spectra of 3 corroborate the solid-state and UV-visible studies. The 1H NMR spectra also lead to observations in accordance with the conclusions from other spectroscopies. The electrochemical behavior of 1 and 2 in acetonitrile or in water solutions shows that the first reduction (Cu(II)Cu(II)-Cu(II)Cu(I) redox couple) is reversible and the second (formation of Cu(I)Cu(I) irreversible. In water, 1 and 2 are reversibly interconverted upon acid/base titration (pK 4.95). In basic medium a new species, 4, is reversibly formed (pK 12.0), identified as the bishydroxo complex. Only 1 exhibits catecholase activity (oxidation of 3,5-di-tert-butylcatechol to the corresponding quinone, vmax = 1.1 x 10(-6) M-1 s-1 and KM = 1.49 mM). The results indicate that the pH dependence of the catalytic abilities of the complexes is related to changes in the coordination sphere of the metal centers.

Calcein as a fluorescent probe for ferric iron. Application to iron nutrition in plant cells.

The recent use of calcein (CA) as a fluorescent probe for cellular iron has been shown to reflect the nutritional status of iron in mammalian cells (Breuer, W., Epsztejn, S., and Cabantchik, Z. I. (1995) J. Biol. Chem. 270, 24209-24215). CA was claimed to be a chemosensor for iron(II), to measure the labile iron pool and the concentration of cellular free iron(II). We first study here the thermodynamic and kinetic properties of iron binding by CA. Chelation of a first iron(III) involves one aminodiacetic arm and a phenol. The overall stability constant log beta111 of FeIIICAH is 33. 9. The free metal ion concentration is pFeIII = 20.3. A (FeIII)2 CA complex can be formed. A reversible iron(III) exchange from FeIIICAH to citrate and nitrilotriacetic acid is evidenced when these ligands are present in large excess. The kinetics of iron(III) exchange by CA is compatible with metabolic studies. The low reduction potential of FeIIICAH shows that the ferric form is highly stabilized. CA fluorescence is quenched by 85% after FeIII chelation but by only 20% using FeII. Real time iron nutrition by Arabidopsis thaliana cells has been measured by fluorimetry, and the iron buffer FeIIICAH + CA was used as source of iron. As a siderophore, FeIIICAH promotes cell growth and regreening of iron-deficient cells more rapidly than FeIIIEDTA. We conclude that CA is a good chemosensor for iron(III) in cells and biological fluids, but not for Fe(II). We discuss the interest of quantifying iron buffers in biochemical studies of iron, in vitro as well as in cells.

Combined intensive chemoradiotherapy for organ preservation in patients with resectable and non-resectable oesophageal cancer.

From January 1990 to April 1993, 60 oesophageal cancer patients were enrolled in a protocol of non-surgical treatment that consisted of induction chemotherapy followed by concurrent chemoradiotherapy. Induction chemotherapy consisted of cisplatin 40 mg/m2 intravenous bolus days 1, 2, 14, 15; 24 h continuous infusion of 5-fluorouracil (5-FU) 1000 mg/m2 days 1 and 14; leucovorin 20 mg/m2 days 1 and 14 given before and with 5-FU; bleomycin 30 UI days 1 and 14; mitomycin C 10 mg/m2 day 14. Concurrent chemoradiotherapy consisted of 60 Gy (6 weeks) from day 21 and cisplatin 70 mg/m2 days 28, 42 and 56; leucovorin 20 mg/m2 followed by 5-FU 425 mg/m2 days 28, 35, 42, 49 and 56. Complete response occurred in 44 of 55 evaluable patients (80%). The median survival is 32 months; the actuarial survival at 40 months is 35% (CI 18-53). These results appear improved over those reported with surgery or radiation alone, and suggest that organ preservation as a secondary treatment goal should be vigorously investigated.

Thyroglobulin autoantibodies in endocrine diseases.

A simple, rapid haemagglutination laboratory kit was developed for the measurement of the thyroglobulin autoantibodies (Tgl-AAb). The Tgl-AAb kit was applied to the measurement of the AAbs titres in the sera collected from 2861 endocrine patients either hospitalized in the Institute of Endocrinology (about 75%) or endocrine outpatients--the great part of patients (over 90%) being however diagnosed as thyroid disorders. The sex classification showed the F/M ratio 6.61/1 for the whole group and 7.4/1 for the positive cases. The prevalence of the Tgl-AAbs positive sera at low titre (under 1:200) is 11.6% about twofold higher than in a population of 700 blood donors (6.4%), previously reported. Among the significant Tgl-AAbs titres, chosen more or less arbitrarily over 1:200, 30.47% of the patients have titres in the range 1:200-1:5000. The Tgl-AAbs positive and negative sera are classified and analysed according to the endocrine pathology but especially thyroid disorders and the positive thyroid disease are grouped by low, high and very high Tgl-AAbs titres and by Tgl-precipitin positive. Our results are rather similar to those reported for endocrine collectivities in other countries. The technical and methodological sources of some discrepancies between the laboratory Tgl-AAbs results and the clinical symptoms and/or anatomo-pathologic results are commented as well as some of the future perspectives of the laboratory investigation of the thyroid autoimmune diseases. Concluding, a strategy schema for a possible immunogram is presented.

A radioimmunoassay system for LH based on the antigenic similitude of LH with HCG: methodology and applications.

A double antibody radioimmunoassay (RIA) system for LH (LH-IEP Kit) was developed using the antigenic similitude of LH with HCG. The first antibody (Ist Ab) is rabbit anti-HCG serum, initial dilution 1:200 000. The tracer is 125I-HCG (code MJ-14 Swierk Poland). The standard curve is calibrated with the reference preparation hLH-Ist-IRP 68/40 kindly offered by WHO. The IInd Ab is pig anti-rabbit IgG serum. The incubation conditions: volume-0.3 ml; time-24 hrs with Ist Ab and 24 hrs with the IInd Ab at ambient temperature. The sensitivity of the RIA system for LH is 1.5 mIU/ml. To validate our RIA system the LH was measured in the serum samples collected from 9 women during the menstrual cycles, from 2 boys during the GnRH test, from 2 amenorrheic women and from 20 children, adolescents and adults with miscellaneous pathologies. In all these samples, parallel measurements of LH and FSH were performed using DDR commercial RIA Kits-SSW. It is to be mentioned that the LH-RIA Kit-SSW is not completely homologous, the Ist Ab being rabbit serum anti-HCG. The results obtained during 4 menstrual cycles, in which the LH peak is observed around the mid point of the interval are: follicular phase 17.92 +/- 5.58 mIU/ml (means +/- SD) with LH-IEP-Kit and 5.51 +/- 2.77 mIU/ml with the LH-SSW-Kit, peak: 26.07 +/- 22.13 mIU/ml and 9.13 +/- 5.60 mIU/ml respectively; luteal phase: 12.55 +/- 5.46 mIU/ml and 3.4 +/- 2.38 mIU/ml, respectively. The LH values observed by the two kits through all 9 menstrual cycles are well correlated ("r" values in the range 0.7-0.9) but high discrepancies were observed in the remaining 3 cycles ("r" between 0.07 and 0.6). These discrepancies as well as those observed in some adolescents with genetic anomalies and in a patient at climacterium are suggesting that the two LH-RIA systems measure not only a common molecular area but also different areas of the LH circulating molecules.

Peptide hormones in saliva. I. Insulin in saliva during the oral glucose tolerance test in female patients.

The radioimmunoassay (RIA) of insulin was performed in the serum and saliva of 27 female patients during the oral glucose tolerance test (OGTT). The patients were divided into two groups: 19 non-diabetic patients and 8 patients diagnosed as impaired glucose tolerance (IGT) disease. In one patient in each group, the OGTT was performed twice at intervals of 3-5 days. The results show that immunoreactive insulin (IRI) is present in saliva and its concentration increases during the glucose stimulation test from 6.48 +/- 1.13 microU/ml (means +/- SEM) in basal conditions at peak values of 45.46 +/- 10.14 microU/ml at 2 hrs after glucose intake. In patients with IGT salivary IRI increases from 5.18 +/- 1.39 microU/ml in basal conditions to peak values of 83.34 +/- 25.85 microU/ml at 3 hrs after glucose administration. Great response variations were observed either inter-individual or intraindividual in both groups of patients. Some patients had unusual high salivary IRI concentration especially in those with gastrointestinal troubles. Further, some hypotheses and experimental models, are advanced, considered useful for the explanation of the physiologic significance of the salivary IRI or of the IRI-like material.

Radioreceptorassay of insulin using human erythrocytes. I. Methodological aspects.

It is known that human erythrocytes have specific receptors for insulin. This works describes a radioreceptor assay (RRA) suitable for the determination of the insulin binding to the surface receptors of erythrocytes. The erythrocytes were isolated from heparinated blood collected in the morning after overnight fasting. In the incubation mixture are 4-5 X 10(9) cells/ml, labelled porcine insulin and nonlabeled (cold) insulin in serial concentrations, range 10-10(5) ng/ml. Incubation (150 min. at +15 degrees C) is ended by suspending the incubation mixture in cold buffer. After washing, the tracer insulin bound to erythrocytes is measured on a gamma counter and the percentage of the specific binding is calculated against the total radioactivity determined prior to the washing steps. By this procedure, the maximal binding of 125I-Insulin was 10.55 +/- 0.78% (mean +/- SD) in five normal volunteers and 6.79 +/- 1.77 (mean +/- SD) in five obese nondiabetic patients, having an enhanced insulin response in the oral glucose tolerance test. The slight hemolysis that happened accidentally in some tubes after incubation caused rude degradation of the tracer, the binding being decreased at last by 25%. The method described (according to Gambhir K. K. et al 1977 slightly modified) is specific and thus suitable for estimating the prognosis of obese children, patients with acute and chronic hepatitis and also for the study of the role of erythrocytes in insulin metabolism and possibly in the metabolism of other hormones.

The entrapment of biological active substances into liposomes. II. Effects of oral administration of liposomally entrapped insulin in normal and alloxanized rats.

Insulin (I) was entrapped into liposomes of various lipid compositions. After oral administration, some of the normal rats treated with neutral liposomes (c) and (d) having egg yolk lecithin, prepared with 2 or 4 mg I/ml respectively, showed a decrease in blood glucose at 2 and 4 hrs. In alloxanized rats (75 mg alloxan/kg body weight) the positive liposomes (f) have induced in some animals a decrease in blood glucose at both time intervals after oral treatment. When neutral liposomes (c) were administered to diabetic rats (125 mg alloxan/kg body) the blood glucose decreased in 9 of 12 animals from 391 to 125 mg/100 ml blood, at 3 hrs after oral treatment. At 1 h, the effect had appeared in 3 of 14 animals. The neutral liposomes (g) having synthetic lecithin, induced a decrease in blood glucose in 5 of 10 animals. A good correlation was observed between decreased glucose and increased insulin levels with the same variability throughout the treated group. No increased insulin levels were observed in diabetic control and normal rats. By the RIA method, the insulin entrapment was 8%. Histological studies have shown that at 35 min after oral administration, neutral liposomes (c) penetrate deeply into the intestinal wall of alloxanized rats as compared both to the neutral liposomes with synthetic lecithin (g) or to the positive ones (f).

A radioimmunoassay system for human myoglobin: method development and applications.

double antibody radioimmunoassay (RIA) system for human myoglobin (hMb) was developed using our own reagents. The antigen (hMb) was isolated from human muscle, purified and stored frozen until needed for immunization, radiolabeling or reference preparation. The anti-hMb serum raised in rabbits was used at 1:2.10(4) dilution (initial). The Chloramine-T method was used for the hMb labeling obtaining at 10-15 muCi/micrograms (370-550 KBq/micrograms) specific activity. Working standards were prepared having concentrations in the range of 2.0 to 500 ng/ml. The reagents were incubated at +4 degrees C for 48 plus 24 hrs. The specificity and accuracy of our hMb-RIA system were validated using for parallel assays an already validated immunochemical system, the hemagglutination inhibition (HI) technique and the parallelism test using serum dilutions from patients with acute myocardial infarction (AMI). The serum hMb concentration in normal subjects (no = 23) was 54.14 +/- 15.08 ng/ml (X +/- SD), being higher in short-term hypothyroidism (no = 13), 87.95 +/- 20.90 ng/ml (p less than 0.0005) or in treated hyperthyroidism (no = 5), 80.03 +/- 21.81 ng/ml. In AMI (no = 6) the serum hMb concentration varied in the range of 123 to 1510 ng/ml. The sensitivity of our hMb-RIA system is 2 ng/ml and the intraassay average error (coefficient of variability % in %B) is 2.26%. Trials to shorten the incubation time showed that adequate binding of labelled Mb may be obtained with 2 plus 4 hr intervals at room temperature. It is necessary to establish, in our conditions, the variation limits for serum hMb in normal subjects according to sex and age as a comparison basis for the study of its physiological and pathological variations.

Immunohistochemical demonstration of thyroglobulin in thyroid pathology.

Thyroglobulin (Tg) was detected by the immunoperoxidase method in the following thyroid diseases: euthyroid goiter, Graves' disease, Hashimoto's thyroiditis, folliculo-papillary carcinoma, follicular carcinoma, anaplastic carcinoma and medullary carcinoma. Thyroglobulin was present in all benign lesions. The highest immunohistochemical staining reaction was found in Graves' disease. In euthyroid goiter, some colloid-distended macrofollicles did not show any Tg staining. A heterogeneous pattern of Tg staining was displayed especially by thyroid carcinomas. In most cases of papillary and follicular carcinomas Tg was detected in some neoplastic cells and sometimes in the colloid. There were differences in Tg content between different fields of the same tumor. In all anaplastic carcinomas a negative Tg staining was found. Vizualization of Tg in thyroid carcinomas is important for estimating the degree of differentiation and for indicating the thyroidal origin of metastases of adenocarcinomas.

The simultaneous isolation of the human pituitary hormones. II. The luteinizing hormone.

The main purpose of the work is the isolation of luteinizing hormone (LH) from frozen human pituitaries. The procedure for the processing of the glands included the steps described by P. Roos [22] for the preparation of the follicle stimulating hormone (FSH) slightly modified and adapted to our conditions. After thawing, the pituitaries were homogenized in 0.03 N Naphosphate buffer, pH 5.7. The protein bulk containing growth hormone (GH) was separated by precipitation with saturated ammonium sulfate (v/v). The supernatant containing gonadotrophins, obtained by centrifugation, was fractionated by chromatography on DEAE cellulose, the first peak being further fractionated by column gel-filtration on Sephadex G-100 after a previous concentration by dialysis against polyvinylpyrolidone 10%. The first peak obtained by this last fractionation was considered for the time being, as pure LH. The extraction and fractionation steps were followed by disc polyacrylamide gel electrophoresis and by radioimmunoassay measurement of the LH concentration. Studies concerning the homogeneity and the biological activity of our LH preparation by comparison to the LH preparations recommended by WHO, are in progress in our laboratory.

An improved procedure for the preparation of the human thyroglobulin and the development of a thyroglobulin autoantibody kit.

Human thyroglobulin has various applications as a diagnosis reagent or for studying the physiopathology of the protein and hormonal biosynthesis in the thyroid gland. The previous preparation procedure for isolating the human thyroglobulin currently used in our laboratory, has some inconveniences as regards the low yield and the noxious influence of the ammonium sulfate precipitation upon its molecular integrity. Therefore, we describe an improved fractionation and purification procedure whose main steps are extraction of the thyroid tissue homogenate in 0.15 M NaCl followed by a double gel filtration on Sephadex G-200 column. In this way are obtained thyroglobulins A and B grade using the two chromatographic steps, respectively. Thyroglobulin B grade is further submitted to preparative polyacrylamide gel electrophoresis for separating some thyroglobulin isomers as recognized by other groups using the analytical ultracentrifugation procedure. The different fractionation and purification steps were checked by double diffusion in gel using rabbit anti-thyroglobulin serum and horse antihuman serum protein. The homogeneity and the molecular weight of the different fractions we evidenced were analyzed by the aid of disc and plate electrophoresis in polyacrylamide gel. The authors developed the technique for thyroglobulin autoantibody detection by the passive haemaglutination method using stabilized erythrocytes coated with thyroglobulin A-grade. Thyroglobulin B-grade used as a tracer and a reference preparation in a RIA system offered a sensitivity of 1.5 micrograms/liter for thyroglobulin detection in biological fluids.

The simultaneous isolation of human pituitary hormones. I. Human growth hormone.

The main purposes of the present work are: a. the preparation of "clinical grade" human growth hormone (hGH), its physico-chemical analysis and the improvement of its solubility for clinical purposes; b. the development of a method for the isolation of high-purity hGH using frozen pituitaries. Nine batches of 20 g acetone powder were processed resulting in 4940 mg of "clinical grade" hGH. Samples of these batches randomly selected were analysed by Sephadex G-100 chromatography and by disc and preparative polyacrylamide gel electrophoresis (PAGE). Lyophilised hGH, soluble in NaCl 0.15 M was prepared and called "Hormcresc" and directions for use were elaborated. One hundred frozen glands were processed and the "crude" hGH was purified by gel filtration on Sephadex G-100 and tested using double diffusion in agar gel, radioimmunoassay (RIA), rechromatography on Sephadex G-100 and disc PAGE. The experiments led to an extraction yield of 550 +/- 165 (means +/- SD) mg "clinical grade" hGH per 20 g of acetone powder. The elution pattern of Sephadex G-100 chromatography and of preparative PAGE as well as the pattern of disc PAGE showed that the "clinical grade" hGH is similar to the already known GH hormones: Raben Somatrotropin, Crescormon (Sweden) and hGH (FRG) but different from Sotropin-H (DDR). The "clinical grade" hGH in lyophilised form is similar to the GH preparations accepted by the European pharmacopoea; it is soluble in NaCl 0.15 M and painless on injection by comparison to hGH in powder form. A method was worked out for the extraction, isolation and purification of "highly pure" hGH using frozen pituitaries, which made it possible to isolate from the same batch of glands not only hGH but also luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin and thyroid stimulating hormone (TSH). During the purification of "crude" hGH on Sephadex G-100 a rather abundant fraction of MW of about 5000-15000 daltons was observed; this fraction, codified provisionally the "X" fraction does not contain hGH, as results from the RIA measurements. On disc electrophoresis, the purified hGH is not homogeneous: behind the main fast band three slower bands are observed. Studies concerning the comparison of our "highly pure" hGH with the hGH preparations recommended by WHO, are in progress in our laboratory.

Insulin radioimmunoassay measurement by the polyethyleneglycol separation technique using the ROTOP kit reagents. Some clinical applications.

The technique described in the instructions booklet of the radioimmunoassay Insulin kit commercially known as the ROTOP-Insulin kit (CCR) tested both in our and in other laboratories did not show the desired reproducibility. Using the main ROTOP reagents (anti-insulin serum and 125I-Insulin) in radioimmunoassay (RIA) systems with other separation techniques (double antibody, dextran-charcoal and polyethyleneglycol) we observed that these reagents are suitable for a sensitive and precise RIA system. The intra-assay variations observed were caused by the separation technique with 86% ethanol as proposed by the procedure. This work describes an insulin-RIA system modified in order to be able to use the ROTOP reagents with the polyethyleneglycol (PEG) separation technique. This variant is used for more than three years in our laboratory but was adopted also in other RIA laboratories in Romania.

A radioimmunoassay system for human thyroglobulin.

A radioimmunoassay (RIA) system for thyroglobulin (Tgl) was developed. The crude Tgl was prepared from thyroid glands surgically removed for colloid goiter by ammonium sulphate fractionation. Two purified Tgls were obtained by gel filtration on Sephadex G-200 (Tgl-200) and by preparative polyacrylamide gel electrophoresis (Tgl-PAGE). Both Tgl-200 and Tgl-PAGE were used for immunization of the animals for radioiodination and as reference preparations. The double antibody RIA system using Tgl-200 may detect 1.5 microgram/l, the technique being suitable for Tgl quantification in human biological fluids. The results of the measurement in 54 children without apparent thyroid enlargement showed a rather wide range of variability of the Tgl values in basal conditions, for example 4.67-37.05 micrograms/l in a group of 29 boys aged 6 to 16 years and 4.06-31.90 micrograms/l in a group of 25 girls aged 5 to 16 years.