Analysis of the diagnostic presentation profile, parathyroidectomy indication and bone mineral density follow-up of Brazilian patients with primary hyperparathyroidism

Primary hyperparathyroidism is an endocrine disorder with variable clinical expression, frequently presenting as asymptomatic hypercalcemia in Western countries but still predominantly as a symptomatic disease in developing countries. The objective of this retrospective study was to describe the diagnostic presentation profile, parathyroidectomy indication and post-surgical bone mineral density follow-up of patients with primary hyperparathyroidism seen at a university hospital. We found 115 patients (92 women, median age 56 years) with primary hyperparathyroidism diagnosed during the last 20 years. We defined symptomatic patients based on the presence of any classical symptom affecting bone, kidney or the neuromuscular system. Surgical criteria followed the guidelines of the National Institutes of Health regarding asymptomatic primary hyperparathyroidism. Symptomatic patients and patients meeting surgical criteria for parathyroidectomy were 66 and 93 percent of the sample, respectively. Median calcium and parathyroid hormone values were 11.9 mg/dL and 189 pg/mL, respectively. After surgical treatment, 97 percent of patients were cured, with increases in bone mineral density of 19.4 percent in the lumbar spine and 15.7 percent in the femoral neck 3 years after surgery. Greater bone mass increases were detected in pre-menopausal women, men, and in symptomatic and younger patients, both in the lumbar spine and femoral neck. Our results support the previous findings of a predominantly symptomatic disease with a presentation profile that could be mainly related to a delayed diagnosis. Nevertheless, genetic and racial backgrounds, and nutritional factors such as calcium and vitamin D deficiency may play a role in the clinical presentation of primary hyperparathyroidism of Brazilian patients.

Changes in clinical and laboratory findings at the time of diagnosis of primary hyperparathyroidism in a University Hospital in São Paulo from 1985 to 2002

In contrast to most developed countries, most patients with primary hyperparathyroidism in Brazil are still symptomatic at diagnosis. However, we have been observing a change in this pattern, especially in the last few years. We evaluated 104 patients, 77 females and 27 males aged 11-79 years (mean: 54.4 years), diagnosed between 1985 and 2002 at a University Hospital. Diagnosis was made on the basis of clinical findings and of high total and/or ionized calcium levels, high or inappropriate levels of intact parathyroid hormone and of surgical findings in 80 patients. Patients were divided into three groups, i.e., patients diagnosed from 1985 to 1989, patients diagnosed from 1990 to 1994, and patients diagnosed from 1995 to 2002. The number of new cases diagnosed/year increased from 1.8/year in the first group to 6.0/year in the second group and 8.1/year in the third group. The first group comprised 9 patients (mean serum calcium ± SD, 13.6 ± 1.6 mg/dl), 8 of them (88.8 percent) defined as symptomatic. The second group comprised 30 patients (mean calcium ± SD, 12.2 ± 1.63 mg/dl), 22 of them defined as symptomatic (73.3 percent). The third group contained 65 patients (mean calcium 11.7 ± 1.1 mg/dl), 34 of them symptomatic (52.3 percent). Patients from the first group tended to be younger (mean ± SD, 43.0 ± 15 vs 55.1 ± 14.4 and 55.7 ± 17.3 years, respectively) and their mean serum calcium was significantly higher (P < 0.05). All of symptomatic patients independent of group had higher serum calcium levels (12.4 ± 1.53 mg/dl, N = 64) than asymptomatic patients (11.4 ± 1.0 mg/dl, N = 40). Our data showed an increase in the percentage of asymptomatic patients over the years in the number of primary hyperparathyroidism cases diagnosed. This finding may be due to an increased availability of diagnostic methods and/or to an increased awareness about the disease.

Response to an oral calcium load in nephrolithiasis patients with fluctuating parathyroid hormone and ionized calcium levels

The response to an oral calcium load test was assessed in 17 hypercalciuric nephrolithiasis patients who presented elevated parathyroid hormone (PTH) irrespective of the ionized calcium (sCa2+) levels. Blood samples were collected at baseline (0 min) and at 60 and 180 min after 1 g calcium load for serum PTH, total calcium, sCa2+, and 1.25(OH)2D3 determinations. According to the sCa2+ level at baseline, patients were classified as normocalcemic (N = 9) or hypercalcemic (N = 8). Six healthy subjects were also evaluated as controls. Bone mineral density was reduced in 14/17 patients. In the normocalcemic group, mean PTH levels at 0, 60 and 180 min (95 ± 76, 56 ± 40, 57 ± 45 pg/ml, respectively) did not differ from the hypercalcemic group (130 ± 75, 68 ± 35, 80 ± 33 pg/ml) but were significantly higher compared to healthy subjects despite a similar elevation in sCa2+ after 60 and 180 min vs baseline in all 3 groups. Mean total calcium and 1.25(OH)2D3 were similar in the 3 groups. Additionally, we observed that 5 of 9 normocalcemic patients presented a significantly higher concentration-time curve for serum PTH (AUC0',60',180') than the other 4 patients and the healthy subjects, suggesting a primary parathyroid dysfunction. These data suggest that the individual response to an oral calcium load test may be a valuable dynamic tool to disclose a subtle primary hyperparathyroidism in patients with high PTH and fluctuating sCa2+ levels, avoiding repeated measurements of both parameters.

Serum leptin concentration during puberty in healthy nonobese adolescents

Data obtained during the past five years have indicated that there are important age- and gender-based differences in the regulation and action of leptin in humans. To study the physiological changes of leptin during puberty in both sexes, and its relationship with body composition and sexual maturation, we measured leptin concentrations in 175 healthy adolescents (80 girls, 95 boys, 10-18 years of age), representing all pubertal stages. We excluded individuals with a body mass index (BMI) below the 5thor above the 95th percentile relative to age. Serum concentrations of leptin were determined by a monoclonal antibody-based immunofluorimetric assay, developed in our laboratory. Body composition was determined by dual-energy X-ray absorptiometry. Pubertal stage was assigned by physical examination, according to Tanner criteria for breast development in females and genital development in males. Leptin concentration in girls (N = 80) presented a positive linear correlation with age (r = 0.35, P = 0.0012), BMI (r = 0.65, P < 0.0001) and percentfat mass (r = 0.76, P < 0.0001). In boys (N = 95) there was a positive correlation with BMI (r = 0.49, P < 0.0001) and percentfat mass (r = 0.85, P < 0.0001), but a significant negative linear correlation with Tanner stage (r = -0.45, P < 0.0001) and age (r = -0.40, P < 0.0001). The regression equation revealed that percentfat mass and BMI are the best parameters to be used to estimate leptin levels in both sexes. Thus, the normal reference ranges for circulating leptin during adolescence should be constructed according to BMI or percentfat mass to assure a correct evaluation

Usefulness of a rapid immunometric assay for intraoperative parathyroid hormone measurements

Intraoperative parathyroid hormone (IO-PTH) measurements have been proposed to improve operative success rates in primary, secondary and tertiary hyperparathyroidism (PHP, SHP and THP). Thirty-one patients requiring parathyroidectomy were evaluated retrospectively from June 2000 to January 2002. Sixteen had PHP, 7 SHP and 8 THP. Serum samples were taken at times 0 (before resection), 10, 20 and 30 min after resection of each abnormal parathyroid gland. Samples from 28 patients were frozen at -70ºC for subsequent tests, whereas samples from three patients were tested while surgery was being performed. IO-PTH was measured using the Elecsys immunochemiluminometric assay (Roche, Mannheim, Germany). The time necessary to perform the assay was 9 min. All samples had a second measurement taken by a conventional immunofluorimetric method. We considered as cured patients who presented normocalcemia in PHP and THP, and normal levels of PTH in SHP one month after surgery and who remained in this condition throughout the follow-up of 1 to 20 months. When rapid PTH assay was compared with a routine immunofluorimetric assay, excellent correlation was observed (r = 0.959, P < 0.0001). IO-PTH measurement showed a rapid average decline of 78.8 percent in PTH 10 min after adenoma resection in PHP and all patients were cured. SHP patients had an average IO-PTH decrease of 89 percent 30 min after total parathyroidectomy and cure was observed in 85.7 percent. THP showed an average IO-PTH decrease of 91.9 percent, and cure was obtained in 87.5 percent of patients. IO-PTH can be a useful tool that might improve the rate of successful treatment of PHP, SHP and THP

Bone mineral density in young women of the city of Säo Paulo, Brazil: correlation with both collagen type I alpha 1 gene polymorphism and clinical aspects

Osteoporosis is a multifactorial disease with great impact on morbidity and mortality mainly in postmenopausal women. Although it is recognized that factors related to life-style and habits may influence bone mass formation leading to greater or lower bone mass, more than 85 percent of the variation in bone mineral density (BMD) is genetically determined. The collagen type I alpha 1 (COLIA1) gene is a possible risk factor for osteoporosis. We studied a population of 220 young women from the city of Säo Paulo, Brazil, with respect to BMD and its correlation with both COLIA1 genotype and clinical aspects. The distribution of COLIA1 genotype SS, Ss and ss in the population studied was 73.6, 24.1 and 2.3 percent, respectively. No association between these genotypes and femoral or lumbar spine BMD was detected. There was a positive association between lumbar spine BMD and weight (P<0.0001), height (P<0.0156), and body mass index (BMI) (P<0.0156), and a negative association with age at menarche (P<0.0026). There was also a positive association between femoral BMD and weight (P<0.0001), height (P<0.0001), and BMI (P<0.0001), and a negative correlation with family history for osteoporosis (P<0.041). There was no association between the presence of allele s and reduced BMD. We conclude that a family history of osteoporosis and age at menarche are factors that may influence bone mass in our population

Circulating forms of parathyroid hormone detected with an immunofluorometric assay in patients with primary hyperparathyroidism and in hyperparathyroidism secondary to chronic renal failure

In patients with uremia, intact parathyroid hormone (PTH) measurement appears to overestimate the biologically active hormone in circulation. The recent description of the accumulation in these patients of a non-intact PTH form measured by the standard immunometric assays, re-opened the question. In this study we submitted serum samples from 7 patients with primary hyperparathyroidism (PHP) and from 10 patients with hyperparathyroidism secondary to chronic renal failure (SHP) to preparative HPLC in order to discriminate the molecular forms measured by our currently used immunofluorometric assay for intact PTH. The elution profile obtained with the HPLC system showed two clearly defined peaks, the first one corresponding to a lower molecular weight form, and the second to the intact PTH (1-84) form. In patients with SHP the area under the curve for the first peak (mean 29.5 percent, range 20.6 to 40.4 percent) was significantly greater than that observed for patients with PHP (mean 15.6 percent, range 5.6 to 21.9 percent). This confirms previous studies showing accumulation of molecular forms of slightly lower molecular weight, presumably PTH (7-84), in patients with SHP and, to a lesser extent, in patients with PHP. The real necessity of assays that discriminate between these two molecular forms is debatable

Specific insulin and proinsulin secretion in glucokinase-deficient individuals

Glucokinase (GCK) is an enzyme that regulates insulin secretion, keeping glucose levels within a narrow range. Mutations in the glucokinase gene cause a rare form of diabetes called maturity-onset diabetes of the young (MODY). An early onset (less than 25 years), autosomal dominant inheritance and low insulin secretion stimulated by glucose characterize MODY patients. Specific insulin and proinsulin were measured in serum by immunofluorimetric assays (IFMA) during a 75-g oral glucose tolerance test (OGTT). Two kindreds (SA and LZ) were studied and compared to non-diabetic unrelated individuals (control group 1) matched for age and body mass index (BMI). In one kindred, some of these subjects were also obese (BMI>26 kg/m2), and other family members also presented with obesity and/or late-onset NIDDM. The MODY patients were also compared to a group of five of their first-degree relatives with obesity and/or late-onset NIDDM. The proinsulin profile was different in members of the two MODY kindreds. Fasting proinsulin and the proinsulin/insulin ratio were similar in MODY members of kindred LZ and subjects from control group 1, but were significantly lower than in MODY members of kindred SA (P<0.02 and P<0.01, for proinsulin and proinsulin/insulin ratio, respectively). Moreover, MODY members of family SA had higher levels of proinsulin and proinsulin/insulin ratio, although not significantly different, when compared to their first-degree relatives and to subjects from control group 2. In conclusion, we observed variable degrees of proinsulin levels and proinsulin/insulin ratio in MODY members of two different kindreds. The higher values of these parameters found in MODY and non-MODY members of kindred SA is probably related to the obesity and late-onset NIDDM background present in this family.

Hipocalcemia e síndrome convulsiva em alcoólatras: uma associaçäo frequentemente näo diagnosticada / Hypocalcemia and convulsive syndrome in alcoholics: an association frequently not diagnosed

Hipocalcemia é um fator importante contribuinte para a epilepsia, e estudos anteriores mostraram que o etanol diminui o cálcio plasmático. Objetivo. Estabelecer a prevalência de hipocalcemia na populaçao convulsiva em geral e identificar um subgrupo específico de risco para hipocalcemia. Métodos. Realizamos um estudo prospectivo de dosagem de cálcio ionizado plasmático em 78 pacientes consecutivos admitidos no Serviço de Emergência da Escola Paulista de Medicina devido à síndrome convulsiva. Desses pacientes, 22 por cento eram alcóolicas. Foi também dosado o cálcio ionizado plasmático numa populaçao de 44 alcoólatras nao-convulsivos em seguimento ambulatorial. Resultados. Uma alta prevalência de hipocalcemia foi encontrada na populaçao alcoólatra convulsiva (32 por cento), em contraste com os convulsivos nao-alcoólatras e o grupo alcoólatra nao-convulsivo (ambos os grupos sem distúrbio do cálcio). O cálcio ionizado plasmático do grupo de alcoólatras convulsivos teve valores significantemente menores que o grupo de convulsivos nao-alcoólatras (p<0,05) [mediana de 1,20; variaçao entre 1,04 e 1,32 mmoI/L/; mediana de 1,24; variaçao entre 1,16 e 1,29 mmol/L, respectivamente]. O grupo de alcoólatras nao-convulsivos apresentou valores de cálcio ionizado entre 1,17 e 1,32 mmol/L, com mediana de 1,26mmol/L. Estes valores nao diferiram dos obtidos nos convulsivos nao-alcoólatras, mas foram significantmente maiores que os do grupo de alcoólatras convulsivos (p<0,05). Os níveis de paratormônio sérico, funçao hepática, fosfatemia e amilasemia estavam normais em todos os pacientes estudados. Conclusao. Estes estudo mostra a importância da dosagem de cálcio plasmático em pacientes alcoólatras com síndrome convulsiva e sugere que a correçao da hipocalcemia possa ser medida importante a ser adotada nestes casos.

Pituitary glycoprotein hormone alpha-subunit secretion by cirrhotic patients

Secretion of the alpha-subunit of pituitary glycoprotein hormones usually follows the secretion of intact gonadotropins and is increased in gonadal failure and decreased in isolated gonadotropin deficiency. The aim of the present study was to determine the levels of the alpha-subunit in the serum of patients with cirrhosis of the liver and to compare the results obtained for eugonadal cirrhotic patients with those obtained for cirrhotic patients with hypogonadotropic hypogonadism. Forty-seven of 63 patients with cirrhosis (74.6 percent) presented hypogonadism (which was central in 45 cases and primary in 2), 7 were eugonadal, and 9 women were in normal menopause. The serum alpha-subunit was measured by the fluorimetric method using monoclonal antibodies. Cross-reactivity with LH, TSH, FSH and hCG was 6.5, 1.2, 4.3 and 1.1 percent, respectively, with an intra-assay coefficient of variation (CV) of less than 5 percent and an interassay CV of 5 percent, and sensitivity limit of 4 ng/l. The serum alpha-subunit concentration ranged from 36 to 6253 ng/l, with a median of 273 ng/l. The median was 251 ng/l for patients with central hypogonadism and 198 ng/l for eugonadal patients. The correlation between the alpha-subunit and basal LH levels was significant both in the total sample (r = 0.48, P<0.01) and in the cirrhotic patients with central hypogonadism (r = 0.33, P = 0.02). Among men with central hypogonadism there was a negative correlation between alpha-subunit levels and total testosterone levels (r = 0.54, P<0.01) as well as free testosterone levels (r = -0.53, P<0.01). In conclusion, although the alpha-subunit levels are correlated with LH levels, at present they cannot be used as markers for hypogonadism in patients with cirrhosis of the liver

Fractures of the proximal femur: correlation with vitamin D receptor gene polymorphism

Fractures are the feared consequences of osteoporosis and fractures of the proximal femur (FPF) are those that involve the highest morbidity and mortality. Thus far, evaluation of bone mineral density (BMD) is the best way to determine the risk of fracture. Genetic inheritance, in turn, is one of the major determinants of BMD. A correlation between different genotypes of the vitamin D receptor (VDR) and BMD has been recently reported. On this basis, we decided to determine the importance of the determination of VDR genotype in the presence of an osteoporotic FPF in a Brazilian population. We studied three groups: group I consisted of 73 elderly subjects older than 65 years (78.5 + 7.2 years) hospitalized for nonpathological FPF; group II consisted of 50 individuals older than 65 years (72.9 + 5.2 years) without FPF and group III consisted of 98 young normal Brazilian individuals aged 32.6 + 6.6 years (mean+SD). Analysis of VDR gene polymorphism by restriction fragment lenght polymorphism (RFLP) was performed by PCR amplification followed by BsmI digestion of DNA isolated from peripheral leukocytes. The genotype distribution in group I was 20.5 percent BB, 42.5 percent and 37 percent bb did not differ significantly from the values obtained for group II (16 percent BB, 36 percent Bb and 48 percent bb) or for group III (10.2 percent BB, 47.6 percent Bb and 41.8 percent bb). No differences in genotype distribution were observed between sexes or between the young and elderly groups. We conclude that determination of VDR polymorphism is of no practical use for the prediction of FPF. Other nongenetic factors probably start to affect bone mass, the risk to fall and consequently the occurence of osteoporotic fractures with advancing age.

Comparaçäo entre duas estratégias para a detecçäo precoce do hipotireoidismo congênito / Comparison between two strategies for the precocious detection of congenital hypothyroidism

Objetivo. Comparar em recém-nascidos (RN) duas estratégias diferentes para o rastreamento do hipotiroidismo congênito (HC), a dosagem primária de TSH no sangue colhido do cordao umbilical (método 1) e a dosagem primária de T4 no sangue colhido por punçao de calcanhar no 2 dia de internaçao (método 2). Métodos. Os autores compararam as duas estratégias em 10.000 RN. Dosaram o TSH por método imunofluorimétrico sensível em papel de filtro e o T4 por radioimunoensaio em papel de filtro. A coleta de sangue do calcanhar foi realizada no 2 dia de vida. Resultados. Os dois programas diagnosticaram todos os casos de HC nos RN (4 casos, 1/2.500 RN). O índice de rechamada por coleta inadequada foi nulo no método 1 e de 8,5 por cento (850RN) no método 2. O índice de reconvocaçao para confirmaçao de resultados foi de 0,06 por cento (6RN) no método 1 e 2,25 por cento (225 RN) no método 2; quando este método incluía também a dosagem suplementar de TSH, o índice baixou para 1,63 por cento (163 RN). Conclusao. Os dados dos autores evidenciam a superioridade técnica da coleta de sangue a partir do cordao umbilical em relaçao à punçao de calcanhar, assim como da dosagem primária de TSH em relaçao à de T4, uma vez que apresentam índices muito menores de reconvocaçao.

Vitamin D receptor alleles and bone mineral density in a normal premenopausal Brazilian female population

Studies on the association between vitamin D receptor (VDR) polymorphism and bone mineral density (BMD) in different populations have produced conflicting results probably due to ethnic differences in the populations studied. The Brazilian population is characterized by a very broad genetic background and a high degree of miscegenation. Of an initial group of 164, we studied 127 women from the city of Spo Paulo, aged 20 to 47 years (median, 31 years), with normal menses, a normal diet and no history of diseases or use of any medication that could alter BMD. VDR genotype was assessed by PCR amplification followed by BsmI digestion of DNA isolated from peripheral leukocytes. BMD was measured using dual energy X-ray absorpitometry (Lunar DPX) at the lumbar site (L2-L4) and femoral neck. Most of the women (77.6 per cent) were considered to be of predominantly European ancestry (20.6 per cent of them reported also native American ancestry), 12.8 per cent were of African-Brazilian ancestry and 9.6 per cent of Asian ancestry, 41.0 per cent (52) were classified as bb, 48.8 per cent (62) as Bb and 10.2 per cent (13) as BB. The BB, Bb and bb groups did not differ in age, height, weight, body mass index or age at menarche, Lumbar spine BMD was significantly higher in the bb group (1.22 + 0.16 g/cm2) than in the BB group (1.08 + 0.14; P<0.05), and the Bb group presented an intermediate value (1.17 + 0.15). Femoral neck BMD was higher in the bb group (0.99 + 0.11 g/cm2) compared to Bb (0.93 + 0.12) and BB (0.90 + 0.09) (P<0.05). These data indicate that there is a significant correlation between the VDR BsmI genotype and BMD in healthy Brazilian premenopausal females.

Calcitonina monomérica plasmática e hipercalcemia em pacientes portadores de neoplasia pulmonar / Monomeric plasmatic calcitonin and hypercalcemia in lung cancer patients

A calcitonina (CT) é um hormonio peptídico relacionado ao metabolismo de calcio produzido pelas células C da tiróide. Encontra-se com níveis plasmaticos bastante elevados no carcinoma medular de tiróide e mostra-se como excelente marcador dessa doença. No entanto, existem relatos na literatura que demonstraram níveis elevados deste peptídio em pacientes portadores de outras neoplasias, principalmente no carcinoma de pulmao. Objetivo. Avaliar a validade da dosagem da CT sérica como possível marcador tumoral em pacientes portadores de tumor de pulmao de diferentes tipos histológicos. Métodos. Foram dosados CT plasmatica e calcio ionizado sanguíneo em 56 pacientes portadores de tumores malignos de pulmao. Para as dosagens de CT os autores utilizaram um método de radioimunoensaio específico, realizado após extraçao prévia do soro em coluna de sílica. Resultados. Observou-se prevalência de hipercalcemia de 21,4 por cento; apenas três (5,4 por cento) dos 56 pacientes investigados apresentaram níveis pouco elevados de calcitonina, e o restante manteve níveis normais ou identectaveis do peptídio. Conclusao. Os resultados demonstram que, com a utilizaçao de um método bastante específico para dosagem da calcitonina em sua forma monomérica, nao se encontram níveis elevados deste hormonio em pacientes portadores de neoplasia pulmonar, desestimulando sua utilizaçao como marcador tumoral nesta patologia.

A specific and highly sensitive time-resolved fluoroimmunoassay for human proinsulin

We describe a time-resolved fluoroimmunoassay specific for human proinsulin using a combination of two high-affinity monoclonal antibodies, one against insulin and the other specific for intact proinsulin and for split 65-66 and des 64-65 proinsulin forms. The assay employs only 200 micro liters of serum, with a detection limit of 0.1 pmol/l. The intra-assay variation coefficient was less than 3 percent between 3 and 1000 pmol/l. There was 0 percent cross-reaction with insulin, C-peptide, split 32-33 and des 31-32 proinsulin. Serum concentration of proinsulin was analyzed in 50 subjects during an oral glucose tolerance test (10 non-obese controls, 10 obese controls, 10 subjects with impaired glucose tolerance, 10 patients with type II diabetes meIlitus (DM) and fasting blood glucose (FBG) <140 mg/dl, and 10 patients with type II DM and FBG >150 mg/dl). Mean fasting serum proinsulin levels measured by this assay in non-obese controls (0.84 +/-0.90 pmol/l; 0.1-2.4 pmol/l) were lower than the results reported by her investigators. There was an increase of proinsulin related to obesity and increased glucose levels, suggesting that proinsulin levels increase with insulin resistance.

1, 25-Dihydroxyvitamin D3 stimulates growth and inhibits calcitonin secretion in a human C cell carcinoma cell line

1,25-Dihydroxyvitamin D3 (1,25D3), calcitonin (CT) and parathyroid hormone are the major calcium-regulating hormones. In addition, 1,25D3 has been reported to be a modulator of cell growth and differentiation in many tissues. recently, a suppressive effect of 1,25D3 on CT secretion and synthesis in C cells was demonstrated in vivo and also in vitro, but there are no data about in effects on thyroid C cell growth. We investigated the effects of [3H]-1,25D3 on basal and stimulated CT secretion and on [3H]-thymidine incorporation, using a human medullary thyroid carcinoma cell line (TT cells). After a 4-day expossure to 1,25D3, TT cells showed a dose-dependent inhibition of basal CT secretion (64 per cento of value for the control group at 100 nM 1,25D3). calcium (3mM) plus K+ (50mM) greatly increased CT secretion in both the control and vitamin D-treated groups. However, in the cells preincubated with 1,25D3 the stimulated CT levels less than observed in controls. A dose-dependent increase in [3H]-thymidine incorporation (200 per cent of the value for the at 100 nM 1,25D3) and in cell number (150 per cent of the value for the control group at 100 nM 1,25D3 after 72h) was observed in the groups treated with 1,25D3. 24,25D3 had no effect on CT secretion or cell growth compared to the control group. These data show that 1,25D3 decreased basal and Ca2+-stimulated CT secretion, a specialized function of these cells, and stimulated their growth. Hence, in contrast to its effects on other cell lines, 1,25D3 appears to induce a dedifferentiation on TT cell

Monoclonal antibodies specific for the free alpha subunit of glycoprotein hormones and their use in the development of a sensitive immunofluorometric assay

Glicoprotein hormone free alpha subunit has been used as a marker for some pituitary tumors and to study the reactivity of glycoprotein hormone-producing cells under different circunstances. We describe a highly sensitive ans specific immunofluorometric assau for the measurement of serum free alpha subunit levels. The assay is based on a monoclonal antibody, specific for free alpha subunit, bound to microtiter plates. As tracer antibody we employed an europium-labelled free/complexed alpha subunit specific monoclonal antibody. Using overnight incubation and 50µl samples, the least detectable dose was of the order of 4 ng/1. Cross-reactivity with LH, TSH, FSH, and hCG was 6.5, 1.2, 4.3 and 1.1 percent, repectively. Normal adult males showed values ranging from 120 to 790ng/l, not different from normal adult premenopausal females (88 to 604 ng/l). In post-menopausal females, serum concentrations were significantly highler, ranging from 341 to 407 ng/l. In 56 patients with untreated pituitary tumors (18 "non-secreting", 25 GH-producing and 13 prolactin-producing tumors), 10 showed high values, 3 of them from the first group, 3 from the second and 4 from the third. We conclude that this highly sensitive assay can be a valualbe tool for the diagnosis and follow-up of selected patients with pituatary tumors and in other circumstances in which the glycoprotein hormone-producing cells of the pituitary require evaluation

Comparison of the determination of insulin by a monoclonal antibody-based immunofluorometric assay and by radioimmunoassay

This paper describes an immunofluorometric assay (IFMA) for insulin and compares it with the classical radioimmunoassay (RIA). Monoclonal antibodies against insulin were produced and used to develop the IFMA. One, immobilized on microtiter plates, was used for capture, the other, labelled with Europium, was used as tracer antibody. The IFMA presentes sensitivity to an amount of insulin of 3 pmol/1 and acceptable valueus for intra- and interassay error. The IFMA presented superimposable curves for human insulin, Arg65/Gly66-split proinsulin and des-Lys64, Arg65, and no cross-reactivity with human proinsulin, Arg32/Glu33 -split and des-Arg31, Arg32. The RIA showed 100 percent cross-reactivity with human proinsulin, 90 pecent with des-Arg31, Arg32 and 170 percent with des-Lys64, Arg65. The assay were used to measure insulin in 300 serum samples from 50 subjects submitted to an oral glucose tolerance test (OGTT). Twenty were normal, 10 had impaired glucose tolerance and 20 non-insulin-dependent diabetes mellitus. The mean value (ñ SEM) obtained bu IFMA was 166.7 ñ 12.1 pmol/1 and the mean value obtained by RIA was 339.6 ñ 18.6, with a correlacion of r = 0.80 (P0.01). Comparison of basal insulin levels of the different groups of individuals using IFMA or RIA led to the same conclusions. The area under curve showed statistically significant differences only for the comparison between normal lean subjects and individuals with impaired glucose tolerance, when measured by RIA...(au)

Nocturnal urinary growth hormone excretion as a criterion for growth hormone deficiency

Nocturnal urinary growth hormone (U-hGH) levels measured by a sensitive immunoenzymometric assay were compared with hGH levels in serum before and after a clonidine test in healthy children and in children with short stature to determine whether U-hGH measurement is useful for the screening of hGH deficiency. The study was carried out on 19 healthy children (10 prepubertal and 9 pubertal subjects) and on 20 children with short stature, 10 with growth hormone deficiency (hGHD) and 10 with constitutional growth retardation. The diagnosis of hGHD was based on a blunted response to two provocative hGH tests in the appropriate clinical setting. Overnight urinary hGH secretion (mean of 3 collections) was measured by an immunoenzymometric assay. The best discrimination was obtained when the results were expressed as ng/h. Only one individual in the prepubertal group (U-hGH, 0.05 ng/h) and one patient in the growth retardation group (U-hGH, 0.08 ng/h) had a urinary hGH value below the highest value (0.17 ng/h) observed in the growth hormone deficiency group. The coefficient of correlation between urinary hGH in ng/h and post-clonidine peak was 0.50 (P = 0.0015), between urinary hGH in ng/l and post-clonidine peak was 0.48 (P = 0.0025), between urinary hGH in ng/l per hour and post-clonidine peak was 0.47 (P = 0.0027). The highest specificity (0.93), sensitivity (0.90), false negative rate (0.96) and false positive rate (0.82) were obtained when U-hGH was expressed as ng/h per night. Measurement of urinary nocturnal hGH excretion is a useful, simple, noninvasive method for the diagnosis of hGH deficiency. However, the day-to-day variability and wide normal range limit its usefulness in mild forms of hGH insufficiency

An enzymeimmunoassay for the determination of thyrotropin (TSH) in drieb blood on filter paper: application to a screening program for detection of congenital hypothyroidism

An enzymeimmunoassay (EIA) for H-TSH (human thyrotropin) in dried blood on filter paper using an anti-H-TSH conjugate with ß-D-galactosidase and tubes coated with an anti-H-TSH was performed fo the screening program for detection of congenital hypothyroidism. The blood volume needed in this assay was 8.7 µl. The precision was evaluated by coefficients of variance within and between assays: 11.86 percent and 14.36 percent for H-TSH levels of 18.5 µU/ml and 35 µU/ml. A good correlation was observed between H-TSH concentration measured by EIA and RIA (r=0.91)