Study on the innocuity of the hypophyseal human growth hormone.

The hypophyseal human growth hormone (hGH), a Raben type laboratory preparation, was re-evaluated as regards its innocuity for therapeutic use. Besides the usual control tests recommended by the Romanian Pharmacopoeia, the contamination of the hGH for clinical use with acute and slow viruses, was investigated taking into account the withdrawal of this hormone in many developed countries. The contamination was absent both with acute viruses as resulted from hemadsorption on cell cultures and counterimmunoelectrophoresis, and with slow viruses as observed from a two year-follow up of guinea pigs injected intracerebrally with the hGH preparation. Further, the content of the growth hormone itself as well as the contamination degree with other pituitary hormones was examined. The hGH-RIA content was 2.23 +/- 0.13 IU/mg (means +/- SEM), range: 1.38-2.80 IU/mg (1st International Standard hGH 80/505-1982). The prolactin contamination assessed by RIA was 187.34 +/- 37.66 ng/mg hGH, range: 28.44-385.20 ng (International Standard WHO: 80/541). The LH and FSH contamination as quantified by the Isocommerz (DDR) RIA kits was with two orders of magnitude lower than 10 IU-LH/IU-hGH, the upper LH contamination limit considered as acceptable. Moreover, the proportion of the large molecular forms in the lyophilized hGH preparation was investigated by polyacrylamide gel electrophoresis. Corroborating the data obtained by these control tests with our previous experience on 149 pituitary dwarfs treated with this hGH preparation during the interval 1964-1984, resulted minor risks of some dangerous side effects of hGH administration in children with growth hormone deficiency by possible contamination with pathogenic agents or with other disturbing hormones.

Growth deficiency with high circulating growth hormone levels: the insulin stimulation test in 39 children.

Thirty nine patients with abnormal high basal hGH levels were selected and analysed as a part of a retrospective study of the results of 1,500 insulin stimulation tests (IST), applied in children and adolescents with growth deficiency. Their height, weight, and bone age were lower than their corresponding chronological age. Both in girls and in boys groups, responders and nonresponders subgroups were detected as judging by the results of the secretagogue action of insulin on hGH. The hGH basal levels were 43.88 +/- 18.27 microU/ml (X +/- SD) in boys (no = 22) and 56.61 +/- 35.21 microU/ml in girls (no = 17). It is to be noted that the hGH nonresponder group had deeper hypoglycemia at 30 minutes post-insulin injection than the responder group: 53.6 +/- 13.0 mg/100 ml (X +/- SD) vs 66.0 +/- 11.5 mg/100 ml respectively (p less than 0.01). Two siblings, a girl and a boy, had the highest basal and stimulated hGH, either during the IST or starvation. One of them, the boy, during the starvation test, had a paradoxical fall of about two orders of magnitude of the serum hGH 4 hr after basal sample collection. These two siblings are similar to the familial Laron type dwarfism. The possible mechanisms of growth deficiency in children with constant high but variable hGH values are discussed, as well as the aspects concerning the therapeutic ways to improve their linear growth.

The serum osteocalcin levels in adult and aged hypothyroid patients.

Osteocalcin (OC) or the bone protein containing gamma-carboxyglutamic acid (BGP or Gla-P), is a specific and sensitive marker of bone turnover. A radioimmunoassay (RIA) system for human osteocalcin was developed with the sensitivity of 0.5 ng/ml. The osteocalcin was measured in sera from 33 hormonally and/or clinically hypothyroid patients: 12 adult and 21 aged patients. For comparison, blood samples were collected from 14 hormonally hyperthyroid adult patients in whom the OC levels were 16.23 +/- 7.57 ng/ml (mean +/- SD) and from hormonally euthyroid adult patients (previously treated hyperthyroid patients) having OC 9.76 +/- 5.32 ng/ml. Abnormal low OC levels were noted in the hypothyroid adult patients group: 1.04 +/- 0.23 ng/ml by comparison to the hypothyroid aged patients 3.76 +/- 2.38 ng/ml (p less than 0.001). Moreover, great variability of the OC serum levels was observed in the aged group, four patients hormonally eu- or hypothyroid having high OC levels in the range: 13.29-55.45 ng/ml and other three patients although hormonally euthyroid but clinically hypothyroid had low OC levels 0.88-2.27 ng/ml. The abnormalities of the OC levels in hypothyroid adult and aged patients are discussed.

Methods of isolation and purification of bovine and rat osteocalcin.

Measurement of osteocalcin (a bone protein containing gamma-carboxyglutamic acid, GLA-P) in the biological liquids and tissues is of interest for studying the mechanisms of bone diseases. It is also helpful in making a diagnosis and in following up the patients with diseases in which the osseous system is affected. Methods have been worked out for isolation and purification of osteocalcin from bovine and rat femur in order to use the purified substance as a starting material for developing radioimmunoassay (RIA) systems for each of these osteocalcins. The work went through the following stages: the processing of the femur bone, preparation of the 200 microns bone powder, demineralization of the powder, and concentration of the bone extract, purification of the extract by molecular exclusion chromatography on Sephadex G-100 and repurification to the product by ion exchanger chromatography in NaCl linear gradient, on DEAE Sephadex A-25. The purity of the substance obtained was tested by disk electrophoresis in polyacrylamide gel. The bovine and rat osteocalcins obtained had the adequate purity for being used as RIA reagents.

The serum osteocalcin levels in patients with thyroid diseases.

The mechanism of bone, calcium, phosphorus and proteins abnormalities observed in hyperthyroidism is rather complex and as yet not wholly understood. Increased serum osteocalcin was recently reported in hyperthyroid patients and its decrease after 4-8 months of treatment. Osteocalcin was measured by RIA in the sera of 211 women and 18 men with thyroid diseases. The patients were divided into 3 groups according to diagnosis: I. polynodular goitre and subacute thyroiditis (59 women, 5 men); II. Graves' disease (70 women, 3 men) and III. thyroid cancer, after treatment by surgery and 131I (82 women, 10 men). The osteocalcin levels in the sera of these patients were: 2.97 +/- 2.63 ng/ml (mean +/- SD) for the women and 3.56 +/- 2.10 ng/ml for the men in the 1st group; 16.31 +/- 11.34 ng/ml for the women and 12.75 +/- 6.09 ng/ml for the man in the IInd group and, 1.01 +/- 0.60 ng/ml for the women and 0.78 +/- 0.46 ng/ml for the men in the IIIrd group. No differences were found between the osteocalcin concentrations in the hyperthyroid female patients treated with antithyroid drugs (no = 58) and the non-treated hyperthyroid women (no = 12): 16.22 +/- 11.40 ng/ml vs 16.74 +/- +/- 11.53 ng/ml. These data suggest that bone resorption stimulated by endogenous thyroid-hormones is a rather resistant processus, persisting even after 6-8 mos of associated anti-thyroid therapy. Further are analyzed the possible causes of the subnormal osteocalcin levels observed in patients with thyroid cancer treated by surgery and radioisotope, whose suppression therapy was discontinued 2-3 weeks before blood sampling.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioreceptor assay of human prolactin using rabbit mammary receptors.

The purpose of this work is to develop the procedures for the preparation of the reagents suitable for the radioreceptor assay (RRA) of human prolactin (Prl). Human purified Prl (NIAMDD-hPrl-16) was labelled with 125I by the Chloramine-T or alternatively by the lactoperoxidase method. As reference preparation we used Prl isolated from the ethanolic step of the routine procedure for the preparation of human growth hormone (hGH) for clinical purposes. The lactogenic receptors were prepared from the pregnant rabbit mammary gland previously stimulated with insulin, cortisone and dried thyroid extract. The final receptor preparations obtained by ultracentrifugation contained 8.85-39.36 mg protein per ml. The prolactin was measured in the human sera and in our hPrl preparations by a double antibody radioimmunoassay (RIA) system using the NIAMDD reagents. We developed a RRA system for hPrl using rabbit mammary receptor preparations with a protein concentration of about 4 mg/ml. The comparative competition showed the same magnitude of the inhibition of the tracer receptor binding of hPrl and hGH. This interference of hGH makes difficult the assessment of the specificity of the hPrl-RRA system otherwise accountable by the structural and/or biological relationship of the three lactogenic hormones: hPrl, hGH and human placental lactogen (hPL). Studies concerning the preparation of a purified and solubilized rabbit mammary receptor and of an antiserum for it are in progress in our laboratory with the objective to provide a useful tool for the investigation of the lactogenic receptor structure and function relationship.

A heterologous radioimmunoassay system for human osteocalcin. Applications in performance sports girls.

Taking into account the involvement of the measurement of osteocalcin, a protein-containing gamma-carboxyglutamic acid (Gla), in investigating the physiopathology of bone and calcium metabolism, a radioimmunoassay (RIA) method useful in human clinics was developed. The reagents for the RIA of human osteocalcin namely, the radioiodination preparation, the reference preparation and the antiserum were obtained starting from bovine bone on account of the structure similarities between bovine and human osteocalcin. The reagents were incubated at 4 degrees C for 48 h followed by the polyethylene-glycol (PEG) separation technique. The sensitivity of the osteocalcin RIA method is 2.0 ng/ml, the intraassay precision is 3-5% and the interassay precision, 8-12% (coeficient of variation). Osteocalcin was measured in the sera of 3 groups of performance sports girls under basal conditions and being of more or less similar ages: rowing girls (n = 10, mean age 16 yr 6 mos), gymnast girls (n = 13, mean age 14 yr 8 mos) and table tennis girls (n = 9, mean age 16 yr). The same samples were used for the measurement of myoglobin. Osteocalcin quantification showed similar levels in the rowing and the table tennis girls, i.e. 11.91 +/- 3.39 ng/ml (X +/- SD) and 9.50 +/- 4.15 ng/ml, respectively. By contrast, in the gymnast girls, the osteocalcin levels were about 4 times higher (44.31 +/- 7.27 ng/ml) than in the other two groups. Myoglobin measurements showed lower levels in the gymnast group, than in the rowing and the table tennis groups, the latter two having similar levels. The multiplicative increase of osteocalcin in the gymnast girls is interpreted and commented in relation to their age and sexual development.

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.

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.

Comparative study of the effects of lisuride and pimozide on the preovulatory surge of FSH, LH and prolactin in rat.

In experiments conducted on adult female Wistar rats, lisuride hydrogen maleate, injected before the critical period of proestrus (between 11 h-13 h) in a single s.c. dose of 0.125 mg/kg body, significantly elevated the preovulatory gonadotropin surge and slightly, non-significantly lowered that of prolactin (assessed between 15-16 h). Pimozide, a selective antidopaminergic neuroleptic, injected according to the same schedule in a dose of 2.5 mg/kg body, strongly augmented the LH surge and also prolactin secretion. Thus, stimulation of gonadotropin secretion by lisuride cannot be ascribed to its dopaminergic agonistic action, but may be attributed to its antiserotoninic activity or to the action on the presynaptic dopamine receptors of the tuberoinfundibular neurons, reducing their inhibitory influence on the gonadotropin surge. The results plead for the inhibitory role of DA in the LH and prolactin secretion; 5-HT may have a similar action in the preovulatory gonadotropin surge and a stimulatory one in the surge of prolactin.

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.

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.

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.

Methodological aspects concerning radio-immunoassay of blood thyroxine applicable to population screening.

A research methodology concerning introduction of thyroxine radioimmunoassay applicable to screening programs is reported. The reagents necessary for T4 assay were prepared and tested and the technical parameters, the stages and technical characteristics of RIA-T4 in the laboratory were established. The RIA method for T4 was compared with the CPBA method and with the commercially available kits, both on samples taken from experimental animals and on hypo-, hyper- and normo-thyroid subjects. The range of T4 normal values could thus be established for the geographic area of Romania: 7.5-10.5 microgram dl-1, with a mean of 9.1 +/- 2 SD. The reagents were applied by extension to thyroxine assay on dry blood spots. The results are comparable with those of direct serum assay. The value of these assays in screening thyroid function in newborns and in the population with remnant thyreopathic endemic dystrophy is discussed.

Hyperthyroidism and pregnancy. I. Serum TBG level in normal pregnancy.

Methods for preparation and qualitative evaluation of the reagents necessary for direct radioimmunoassay of thyroxin-binding globulin (TBG) in serum are described. The reagents are: purified TBG necessary as antigen in preparing the labelled product and plotting the reference curve; 125I-TBG; anti-TBG rabbit serum. The technical stages characteristic of the RIA system are presented. TBG level determined in normal volunteers (about 80% males and 20% females, young subjects living in Bucharest area) was found to be 15.6 +/- 2.23 mg L-1 (M +/- SD) and the PBI, T4, and T3 values at the onset were normal. In another lot formed only from women TBG was 17.3 +/- 3.50. Assay of 136 pregnant women between the 2nd an 9th month of pregnancy showed values of 22.46 (2nd month) and 32.23 mg L-1(8th month). The shape of the increase curve during pregnancy shows two main stages. The importance of the serum TBG level as an additional criterion in the diagnosis of pregnancy (even in the first weeks of life of the foetus) and as an indicator of the placentar function is discussed.

The inhibitory action of the pineal extract on TRH.

The effects of a single dose and of 5 doses (5 days) of a bovine polypeptide pineal extract (PE) on the pituitary TSH reserves and on the blood level of thyroid hormones were studied on hypothyroid patients. The blood levels of PBI, total T4 (radiocompetitive), free T4, T3 and TSH (RIA) in basal conditions and during the TRH test (i.v. administration of 200 mug TRH), In the presence and absence of the treatment with PE were determined. Thirty minutes following i.m. administration of PE a decrease was noticed in the high level of the basal TSH in all the patients as well as maintenance or even exaggeration of the TRH response. After 5 days of PE administration a decrease was noticed in the basal TSH, with preservation of a strong response of the TSH release after TRH administration. In these patients a slight decrease was observed in PBI, total and free T4 and T3, when their initial level was close to normal. The results show the results show the intervention of the pineal extract by inhibiting the endogenous TRH.