Multipoint incremental motor unit number estimation as an outcome measure in ALS.

BACKGROUND: Improved outcome measures are necessary to reduce sample size and increase power in amyotrophic lateral sclerosis (ALS) clinical trials. Motor unit number estimation (MUNE) is a potentially attractive tool. MUNE methods previously employed in multicenter trials exhibited excessive variability and were prone to artifact. OBJECTIVE: To evaluate a modification of standard incremental MUNE in a multicenter natural history study of subjects with ALS. METHODS: Fifty healthy subjects were evaluated twice and 71 subjects with ALS were studied repeatedly for up to 500 days. Side and nerve studied was based on clinical examination findings. Nerves were stimulated at 3 specified locations and 3 increments were obtained at each location. Average single motor unit action potential (SMUP) amplitude was calculated by adding the amplitude of the third increment at each location and dividing by 9; SMUP was divided into maximum CMAP amplitude to determine the MUNE. RESULTS: Test-retest variability was 9% in normal subjects. Average MUNE for normal subjects was 225 (±87), and was 41.9 (±39) among subjects with ALS at baseline. Subjects with ALS showed clear decrements over time, with an overage rate of decline of approximately 9% per month. SMUP amplitude increased with time in a fashion consistent with the known pathophysiology of ALS. CONCLUSION: Multipoint incremental MUNE has a number of attributes that make it attractive as an outcome measure in ALS and other diseases characterized by motor unit loss. It can be rapidly performed on any EMG machine and has repeatability and rates of decline that favorably compare to other previously described methods.

Hormonal and metabolic response in elite female gymnasts undergoing strenuous training and supplementation with SUPRO Brand Isolated Soy Protein.

BACKGROUND: This study evaluates the metabolic and hormonal response in elite female gymnasts undergoing strenuous training and supplementation with SUPRO Brand Isolated Soy Protein. METHODS. EXPERIMENTAL DESIGN: 14 top female gymnasts (Romanian Olympic Team), took part in a study to examine their hormonal metabolic profile and to investigate any possible changes resulting from a 4-month program of strenuous training and daily supplementation with soy protein at a level of 1 g/kg body weight. Gymnasts wtare randomly assigned to one of two groups seven to the Supplemented Group (A) and seven to the Non-Supplemented Group (B). Both groups took part in the same program, which consisted of strenuous training for 4-6 hours/day (except on Sunday, controlled food intake and supplements including vitamins and minerals. Group A received a supplement of Sports Beverage Protein Mix with SUPRO Isolated Soy Protein (ISP) twice daily. Group B received a placebo identical in appearance and flavour. Selected parameters were measured before and after the 4-month training program (lean body mass, fat mass, serum hemoglobin, protein, fats, urea and creatinine, liver enzymes, serum total calcium and magnesium, immunoglobulins, urinary mucoproteins, serum T3 and T4, estradiol, progesterone, prolactin, testosterone and urinary 17-ketosteroids). RESULTS: Results demonstrated that the Supplemented Group (A) had an increase in lean body mass and serum levels of prolactin (p < 0.01) and T4 and a decrease in serum alkaline phosphetases (p < 0.01). The Non-Supplemented Group (B) had a decreased level of serum T4 and an increased level of urinary mucoproteins (p < 0.05). CONCLUSIONS: Our preliminary conclusions might suggest lower metabolic-hormonal stress in elite female gymnasts undergoing strenuous training and who received daily supplementation with isolated soy protein.

Sandostatin LAR: a promising therapeutic tool in the management of acromegalic patients.

A stable and sustained suppression of growth hormone (GH) secretion was noted in 101 patients treated long term with individual doses (20 and 30 mg in 89 patients, 40 mg in 12 patients) of Sandostatin LAR (Sandoz Pharma Ltd, Basel, Switzerland). Doses of 20 mg and 30 mg at 4-week intervals delivered average octreotide concentrations of 1,348 +/- 483 ng/L and 2,631 +/- 1,026 ng/L, respectively, in steady-state conditions and provided adequate control of patients who had been well controlled during treatment with 0.1 mg and 0.2 mg thrice-daily subcutaneous (SC) Sandostatin. Suppression of GH serum concentrations to less than 5 micrograms, 2 micrograms, and even 1 microgram/L was recorded in more patients and more consistently during long-term treatment with Sandostatin LAR than Sandostatin. A marked decrease or even a normalization of insulin-like growth factor-1 (IGF-1) serum concentrations was observed after the first double-blind 10-, 20-, or 30-mg dose of Sandostatin LAR. A progressive improvement was recorded during long-term treatment, with normalization of IGF-1 serum concentrations in 65.3% of patients. A marked clinical improvement was observed in parallel, with 36 of 101 patients (35.6%) becoming asymptomatic after the nineteenth injection of Sandostatin LAR. A greater than 20% shrinkage of the GH-secreting adenoma was also recorded in 12 of 14 patients treated with Sandostatin LAR after receiving only 2 to 4 weeks of treatment with SC Sandostatin and in 11 of 18 patients receiving Sandostatin LAR as adjuvant therapy after failure of surgery. The systemic tolerability of Sandostatin LAR was good, and most adverse events were mild and short term (1 to 2 days). No impairment of thyroid function was detected. Newly occurring gallstones were recorded in four of 101 patients and microlithiasis in four of 101 after up to 30 months of treatment with Sandostatin LAR. Due to its excellent efficacy, good tolerability, convenience of administration, and acceptability by patients, Sandostatin LAR is considered a promising therapeutic tool in the management of acromegalic patients.

The influence of "selenium organicum" upon the hepatic function of carbon tetrachloride poisoned rats.

The hepatoprotective action of the Romanian preparation Orgasel containing selenium (Se) 5.01 mg/100 g autolysated yeast powder, was tested on adult Wistar rats poisoned with carbon tetrachloride (CCl4). The hepatotoxic agent (a 20% CCl4 solution in oil) was administered i.p. in a single dose of 0.3 ml CCl4 solution/100 g body weight, and the preparation tested (autolysate of seleniated yeast) was administered by gavage in 4 doses (of 100 mg Se powder/100 g animal each) along 2 days. After 48 hrs the animals were sacrificed, then their blood and liver were collected. The treatment with Orgasel significantly reduced the organs, morphological changes (fat liver degeneration, splenomegaly, testicle degeneration) induced by CCl4 poisoning in the rat, an effect found also at the biological parameters levels studied in plasma and liver. In the plasma, the high lipid peroxide concentrations, the increased activity of alkaline phosphatases, and the reduced antioxidative activity generated by CCl4 have been statistically significant brought to the normal range after Orgasel administration. At the liver this treatment significantly decreased the lipid peroxides production, the total lipids and cholesterol concentrations, and statistically significant increased the enzymes activity (alkaline phosphatases, GPT). The results obtained after Orgasel administration proved that this preparation has a global beneficial action upon the organism in the poisoned rat, as well as a strong antioxidative effect, confirming once again the essential role of Se in maintaining cells' integrity.

The development of a radioimmunoassay system for testosterone (T) and dihydrotestosterone (DHT). Part 2. The preparation of antisera to T.

Testosterone-3-O-carboxymethyl-oxime derivative was synthetized and coupled to bovine serum albumin (BSA). The T-3-CMO-BSA conjugate homogenized with Freund's adjuvant used as immunogen was injected multiple sites in rabbits. The antisera collected were characterized in a radioimmunological system, separation with dextran-charcoal using 125I-Testosterone as tracer. The antibody titres varied from one animal to another. The titre of anti-T serum selected for RIA was 1: 10(4)-1: 2 X 10(4) (initial dilution). All anti-T sera 100 percent crossreacted with 5 alpha-dihydrotestosterone but nonsignificant interference was observed with other C19, C21 and C18 steroids. The affinity constant of the selected anti-T serum was in the range 1.4-1.9 X 10(9) litres/mole. The data so far published on the antisera toward testosterone are reviewed. We conclude that the selected anti-T-3-CMO-BSA serum may provide assays for testosterone with potential for clinical applications.

Testosterone in sera of workers exposed to acrylonitrile.

Testosterone was measured through three consecutive years in sera from young and adult male subjects working in a chemical factory exposed to some complex chemical noxae, the major exposure being acrylonitrile (vinylcyanid). In the first yr, (group A), the blood was collected on May 1975 (no 39), the II-nd yr (group B) on March (no 109) and the III-rd yr (group C) on May (no 149). The exposure time varied in each group between 6 mos and 7-10 yrs. For comparison, blood samples were collected from 145 men of comparable age grouped in nonexposed: blood donors (no 37) (group a), new workers (no 23) (group b) and exposed to other chemical noxae in the same factory: Na cyanid (group c, no 23), cyan derivatives (group d, no 22) and pyrolysis (group no 39). The seasonal testosterone variations being considered, the Student's 't' test applied to the hormonal levels in acrylonitrile groups A, B and C showed non significant differences. However, the comparison of the testosterone concentrations in sera of the groups A, B and C vs the control groups investigated during the same month of the year showed much lower levels of the hormone in the first groups (p less than 0.001). These data are suggesting that the exposure to acrylonitrile either by direct participation to the technological chain or by working in the same noxious environment may influence the testosterone synthesis and/or secretion.

The development of a radioimmunoassay system for testosterone (T) and dihydrotestosterone (DHT). Part 3. The preparation of radioiodinated testosterone.

A two step-method was applied for the preparation of a tracer adequate for a radioimmunoassay (RIA) system for testosterone. Histamine was radioiodinated by the Chloramine-T method and then coupled to testosterone-3-carboxymethyl-oxime (T-3-CMO) derivative. After purification by TLC, the steroid tracer was stable in ethanol for at least four months. Using an anti-T-3-CMO-BSA serum, a (bridge) homologous RIA system for testosterone was developed. The reagents (antiserum, tracer and standard) were incubated 2 hrs at 37 degrees C and then the free radioactivity was removed by a dextran-charcoal suspension. The testosterone RIA system, with the sensitivity of 30 pg/tube, is suitable for the measurement of the steroid hormone in biological fluids and tissues.

The development of a radioimmunoassay system for testosterone (T) and dihydrotestosterone (DHT). Part 1. The preparation of the T-derivatives and T-protein conjugates.

Testosterone-17-hemisuceinate (T-17-HS) by esterification with succinic anhydride technique and Testosterone-3-carboxy-methyl-oxyme (T-3-CMO) derivatives were synthesized. The T-protein (BSA) conjugates were prepared by the condensation with carbodiimide technique, for the T-17-HS derivative and by the mixed anhydride technique, for the T-3-CMO. Both the derivatives and the T-BSA conjugates were characterized by melting point, ultraviolet and infrared spectra, thin layer chromatography. The physico-chemical characteristics and the hapten-protein incorporation being convenient: 28 mol T/mol BSA for T-17-HS-BSA and 38 mol T/mol BSA for T-3-CMO-BSA, the T-carrier conjugates are adequate for their use as immunogens.

Growth hormone (GH) release by arginin-vasotocin (AVT) given intranasally in sexually immature children.

Eighteen healthy children were given 50 micrograms synthetic AVT intranasally and samples of serum for GH, magnesium and alpha-amino-nitrogen were drawn 30, 60 and 120 minutes later. There was a significantly different mean of GH peak level (13.1 +/- 3.02 microU/ml) which occurred generally at 30 minutes. The number of GH responders (i.e. peak level greater than 15 microU/ml) was of 7/18. The means of chemical determinations elicited little though opposite changes: slight increases in magnesium and slight decrease in alpha-amino-nitrogen. In a separate day, a sleep test of 30 minutes-duration performed in the morning resulted in a significantly greater GH peak level (28.2 +/- 9.7 microU/ml) but the number of GH responders was nearly the same (6/18). The serum magnesium levels following sleep decreased slightly and unsignificantly. The involvement of serotonergic neurons in mediating GH-releasing effects of AVT is briefly discussed.

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 assessment of the dopaminergic tonus by urinary determinations of homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) in normal, obese and GH-deficient short children.

In order to assess the dopaminergic tonus, urinary determinations of HVA and DOPAC and also of noradrenaline (NA), adrenaline (A), and methoxyhydroxyphenylglycol (MHPG) were performed in 86 obese children, 11 growth hormone (GH)-deficient short children and also in 40 control children. Part of the obese patients were subjected to a low carbohydrate, low calorie diet and also to short-term (9-14 days) courses of diethylpropion (DEP) 50 mg/day, meclofenoxate (MEC) 100 mg/day and thyroid extract (THE) 1-2 mg/kg/day. The GH-deficient patients received only THE in substitutive (5-10 mg/kg/day) doses. Significative correlations between DOPAC and age, weight and height were found in controls. In the obese group a significantly increased mean level of HVA was found (1.45 +/- 0.09 mg/24 h vs 1.15 +/- 0.10 in controls). The excretion of DOPAC was slightly greater but far from significance. There was also a significant decrease of HVA but not DOPAC in the DEP-treated obese. The rest of the drugs and the diet alone were not effective in any way. Normal levels in all metabolites except NA and A were found in GH-deficient short children. The therapy with thyroid extract did not alter the excretion levels. These findings indicate that in infantile obesity the dopaminergic tonus is somewhat increased but its pathophysiological significance is doubtful. In GH-deficient short children of standard appearance the dopaminergic tonus seems to be undistinguishable from normal.

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)

Sleep-induced GH release in otherwise GH deficient children having short stature; a possibly new variant of GH neurosecretory dysfunction (GHND).

Sleep and exercise are considered physiologic stimuli of growth hormone (GH) release from the pituitary. They were used to some extent for the assessment of GH reserve in growth disorders of supposedly pituitary origin. In order to better establish the diagnostic value of the sleep test, we studied 34 children of both sexes aged 6-14 having various degrees of shortness of stature associated with bone age retardation (range of the latter: 3-10 years). Eight healthy controls of the same age were also subjected to the sleep test. The latter was begun at 10:00-11:00 a.m. generally in a dark room under polygraphic control. The subjects were fed a standard breakfast at 8:00-8:30 a.m. After thirty minutes of steep or after reaching the IVth stage of slow-wave sleep, the children were awakened and samples of venous blood for GH determinations were drawn. Two other GH reserve tests were also performed in other days, usually insulin-induced hypoglycemia and glycine intravenous loading. Except sleep, no other GH provocative tests were performed in controls. In normal GH reserve children (21/34) according to other tests, the sleep test was positive (i.e. peak values greater than 15 microU/ml) in 7 cases and the mean GH response was not significantly smaller than the peak recorded after insulin-induced hypoglycemia. In abnormal GH reserve, presumably hypopituitary children (13/34) according to other tests, the sleep confirmed the other tests in 8 cases. In five cases of clinically and endocrinologically "hypopituitary" dwarfism, the sleep test revealed a normal response.(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.

Serum-to-saliva transfer of the immunoreactive insulin (IRI) in children with obesity associated with insulin-resistance.

Assuming that the serum-to-saliva transfer of insulin reflects internalization and re-cycling of the hormone in the membrane-located binding sites of salivary epithelial cells and that these cells have in obesity a'marked decrease in insulin receptor content, it has been postulated that insulin resistance in infantile obesity can be detected by the changes in the salivary immunoreactive insulin during the oral glucose tolerance test (OGTT). The study included 31 obese children and adolescents of both sexes, subjected to OGTT. Samples of blood and saliva were collected at 30, 60, 120, 180 and 240 minutes for determinations of glucose and IRI. The blood glucose values were generally normal whereas IRI was excessively high. The dynamics of salivary IRI was similar (easy peak followed by slow descent) with the mean serum values but lower by about two-thirds, and the peak was 30-60 minutes delayed. The serum IRI values correlated significantly with the saliva ones at all time-intervals except for the 30-minute ones. The serum IRI values were significantly lower at the 30-minute time interval, whereas the salivary IRI were the lowest (and of borderline significance) at the 60-min. time interval. The mean glucose/kg doses given orally were not significantly different in the two groups. It was concluded that a hormonal activity detectable by IRI assay through the PEG separation method does exist, with a concomitant variation of serum-to-saliva transfer as shown by the OGTT test. It was also concluded that since the salivary values are lower, the direction of the flow is from serum to saliva and not the reverse. Finally, on the basis of our data, an "in situ" synthesis of insulin (hormonogenic exocrinism) can not be ruled out.

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.