DNA analysis and parathyroid pathology.

The nuclear DNA content of 85 parathyroid glands (4 carcinomas, 39 adenomas, 21 secondary parathyroid hyperplasias, and 21 normal parathyroid glands) were determined by flow cytometric analysis. All normal parathyroid glands, 85% of the adenomas, and 83.3% of the secondary hyperplastic glands had DNA indices within values of 0.85-1.1. Paraffin-embedded fixed glands showed less DNA staining than that found with fresh or normal glands. Glands from patients with carcinoma showed DNA indices outside the normal DNA index range. When the percent of nuclei within the G0/G1 phase of the cell cycle was compared between the study groups, highly significant results were found. While patients with secondary hyperplasia showed a similar distribution to the normal glands studied, only 48% of primary adenomas showed over 80% of cells within the G0/G1 region. A clear subgroup of adenomas was defined with more rapidly cycling tetraploid cells, and showing classical adenoma pathology. This group showed negative correlation with gland weight, plasma calcium, and ionized calcium. These findings suggested that a different etiology of the disease process occurs between secondary hyperplasia and parathyroid adenoma. Such abnormal adenomas may form a group worthy of long-term follow-up.

Immunohistochemical localization of parathyroid hormone-related protein in parathyroid adenoma and hyperplasia.

Parathyroid hormone-related protein (PTHrP) is invoked as the cause of humoral hypercalcaemia of malignancy (HHM); it is contained in the keratinocyte layer of normal skin; and there is evidence that is is produced by fetal parathyroids. Antibodies against synthetic PTHrP peptides have been raised in rabbits and sheep. This immunohistochemical study has found that primary parathyroid adenomata and hyperplastic glands from patients with chronic renal failure stain positively with antisera against PTHrP(1-34) and PTHrP(50-69). Primary hyperplastic glands are negative. No staining with anti-PTHrP(106-141) antiserum could be detected immunohistochemically in any of the parathyroid adenomata or hyperplasia.

Manejo quirurgico del hiperparatiroidismo / Surgical managemento of hyperparathyroidism

El surgimiento de tecnologias de diagnostico por el laboratorio, cada vez mas sensibles y automatizadas, ha permitido establecer que la incidencia de hiperparatiroidismo (en un 80% de los casos a consecuencia de adenomas paratiroideos) es mucho mayor de lo que se suponia. Aunque la localizacion anatomica de un adenoma paratiroideo con miras a su remocion quirurgica puede lograrse en algunos casos por procedimientos no invasivos, el metodo mas eficaz es la identificacion visual por un cirujano experimentado en el curso de la intervencion quirurgica. Una exploracion paratiroidea en un paciente con hiperpartiroidismo solo debe emprenderse cuando el diagnostico haya sido claramente establecido por determinaciones bioquimicas, incluyendo hipercalcemia en no menso de tres determinaciones. El cirujano debe conocer en profundidad la embriologia y la anatomia normal y anormal de las glandulas paratiroideas, al igual que las implicaciones clinicas y fisiologicas derivadas del papel que junto con la vitamina D y la calcitonina desempena la paratohormona en la homeostasia del calcio

Microfluorometric measurements of cytoplasmic calcium in chief and oxyphil parathyroid cells of adenomatous and hyperplastic glands and of normal-sized glands associated with adenomas.

The effects of extracellular calcium on the cytoplasmic Ca2+ concentration (Ca2+i) were studied by dual-wavelength microfluorometry in individual human parathyroid cells obtained from adenomatous glands and normal-sized glands associated with adenomas in hypercalcemic hyperparathyroidism (HPT), as well as from enlarged glands of patients with uremia with HPT. In comparison with the normal parathyroid tissue, chief cells of the adenomatous and hyperplastic glands showed significantly lower Ca2+, and also right-shifted responses of Ca2+i to increases in the extracellular calcium concentration within the 0.5 to 3.0 mmol/L range. This pathophysiologic disturbance apparently was independent of the cell size. Oxyphil cells of nodules from the hyperplastic glands had lower Ca2+i and responded less to increments in extracellular Ca2+ than the chief cells from the surrounding parts of the same glands. Also the chief cells from the normal-sized glands associated with single adenomas exhibited a disturbance of the regulation of Ca2+i, which was less pronounced than that in the cells of the adenomas. These findings support the presence of relative calcium insensitivity of Ca2+i in chief and oxyphil parathyroid cells from adenomatous and hyperplastic glands. This derangement may also be found in all parathyroid glands of individuals with adenomatous HPT.

Immunocytochemical localization of vitamin D-dependent calcium-binding protein (calbindin) in thyroid parafollicular cells of guinea pig.

The presence of the 28K vitamin D-dependent, calcium-binding protein (28K calbindin) was investigated by immunocytochemistry in normal thyroid glands and parathyroid glands of rats, guinea pigs, rabbits and men, as well as in human thyroid medullary carcinomas and human parathyroid adenomas. In addition, thyroid glands and parathyroid glands of rats and guinea pigs were studied after treatment with vitamin D3 injected intramuscularly at a total dose of 1.2 x 10(6) IU per 100 g body weight. 28K calbindin was found exclusively in parafollicular cells of guinea pigs and never in those of other species investigated. It was present predominantly in the cytoplasm and in lower concentration in nuclei. After vitamin D3 treatment, increased immunoreactivity of 28K calbindin was observed in the cytoplasm and, even more pronounced, in the nuclei. In normal parathyroid cells and in parathyroid tumors and medullary thyroid carcinomas, 28K calbindin was not demonstrable. Our findings suggest an important function of calbindin in the cellular calcium processing of parafollicular cells of guinea pigs.

ABO blood group antigens in parathyroid adenoma and hyperplasia.

Immunohistochemical determination of ABO blood group antigens was performed on parathyroid tissue to see if the presence or absence of such antigens could be used as an aid to distinguish adenoma from hyperplasia in primary hyperparathyroidism. Material from nine cases of solitary adenoma and seven cases of hyperplasia fixed in formalin and embedded in paraffin was studied using monoclonal antibodies and avidin-biotin-peroxidase complex technique. The two categories of tissue did not show any consistent differences in the extent or intensity of immunoreactivity, and the method tested did not permit distinction between adenomatous and hyperplastic disease of the parathyroid glands.

A case of multiple endocrine neoplasia (MEN) type 1; the immunohistochemical and ultrastructural studies of its tumors and the analysis of hormones in tumor extracts.

We reported a case of sporadic multiple endocrine neoplasia type 1, with multiple insulinoma, parathyroid adenoma, and pituitary tumor. Measurement of hormone contents and immunohistochemical studies of the pancreatic tumors showed that the tumors contained insulin, glucagon, somatostatin, and pancreatic polypeptide. Furthermore, the concentrations of these hormones were different in each tumor. Insulin extracted from the pancreatic tumors analyzed by reversed-phase high performance liquid chromatography revealed no structural abnormalities. On the other hand, in gel filtration evaluation of the extract of the parathyroid adenoma, it was found that the tumor extract contained a macromolecular parathyroid hormone (molecular weight 20,000 to 25,000).

A novel parathyroid hormone-related gene product.

A parathyroid hormone-related protein (PTHrP) has been invoked as being responsible for the humoral hypercalcemia of malignancy. Eight of the first 13 amino acids of PTHrP are identical with those in PTH, but there is no other significant homology. The PTHrP gene is located on chromosome 12, whereas that for PTH is on chromosome 11, and the two genes are probably related by a duplication process. Antisera against PTHrP(1-34), which cross-react poorly or not at all with PTH, and antisera against other parts of PTHrP not homologous to PTH were used in immunocytochemistry using a peroxidase-antiperoxidase method, to identify PTHrP in the cytoplasm of cells in a series of unselected parathyroid adenomata. The study was based on our evidence that PTHrP is produced by fetal parathyroid and may be the predominant calcium-regulating hormone in the mammalian fetus. Glands from five patients with parathyroid hyperplasia secondary to chronic renal failure also stained positively for PTHrP. No evidence was obtained for PTHrP in sections from five patients with primary parathyroid chief cell hyperplasia or in a small group of patients with the multiple endocrine neoplasia type 1 or type 2 syndromes.

S-100 protein and neuron-specific enolase in parathyroid glands and C-cells of the thyroid.

Normal parathyroid glands and parafollicular cells (C-cells) of man, rat and rabbit, and also human parathyroid adenomas and medullary carcinomas were investigated for the presence of S-100 protein and neuron-specific enolase (NSE). For determination of the proteins immunoperoxidase methods were applied, i.e., the PAP method and the avidin-biotin system. The antisera, of polyclonal origin, were specifically directed against cow S-100 protein and rat or bovine NSE. The respective antisera are known to crossreact with S-100 protein from man, rat, and rabbit, as well as with NSE from man and rat. Surprisingly, the test for S-100 protein was found to be strongly positive in the parathyroid glands of rat and rabbit and was focally positive in normal and adenomatous human parathyroid glands, but completely negative in C-cells and medullary carcinoma cells. NSE was present in C-cells of rat and man, and in medullary carcinoma cells, but was absent in normal and adenomatous parathyroid cells. The results support data that indicate that both parathyroid cells and C-cells are derived from elements of the neural crest, but undergo different maturation processes during embryological development.

[Abnormal concentration of aluminium in the lysosomes of a primary parathyroid adenoma]. / Concentration anormale d'aluminium dans les lysosomes d'un adénome parathyroïdien primitif.

A primitive parathyroid adenoma has been studied by electron microscopy, analytical ion microscopy and electron probe X ray analysis. A number of lysosomal structures has been observed in the cells. Observation of unstained ultrathin sections shows that these lysosomes contain two varieties of structures: dense homogeneous droplets and very dense and small granulations. Aluminium associated with phosphorus has been detected in high concentration in the small granulations. The relations between aluminium and parathyroid function and the possible role of aluminium in the pathology of the parathyroid gland remain to be clarified.

Hyperparathyroid glands contain G-17 and G-34 gastrin.

To determine if gastrin in hyperparathyroid glands is true gastrin or artifact and to determine the frequency of gastrin in parathyroid glands, 20 parathyroid glands from 11 patients with hyperparathyroidism but without MEA were extracted and analyzed for gastrin. The parathyroid glands from 4 out of 11 patients had measurable gastrin immunoreactivity (10.7 + 6 pg/mg tissue). Column separation chromatography confirmed that this was true gastrin (40% G-34; 50% G-17). Immunohistochemistry with ABC (avidin biotin complex) immunoperoxidase confirmed the presence of gastrin in cytoplasmic vesicles in scattered parathyroid cells. True gastrin does exist in some cells in some patients with hyperparathyroidism.

Epidermal growth factor receptors in parathyroid tumors.

Hyperparathyroidism is caused by parathyroid adenomas, hyperplastic parathyroid glands, or rarely parathyroid carcinoma. Membrane receptors to epidermal growth factor (EGF), a growth-stimulating polypeptide, have been shown in other endocrine tissues such as thyroid, breast, and ovary, but not in parathyroid glands. Therefore we studied abnormal parathyroid glands from fourteen patients for the presence of EGF receptors. The binding of radioiodine-labeled EGF to the crude membrane fractions was studied using competitive inhibition with unlabeled EGF. In ten patients with solitary parathyroid adenomas, seven adenomas had no EGF binding, three had low affinity EGF binding with dissociation constants (Kd) of 28 to 148 nM and maximal specific binding (Bmax) of 285 to 1944 fmole/mg protein. In two patients with multiple adenomas, a high affinity EGF binding with Kd of 0.28 to 2.8 nM and Bmax of 6.7 to 43 fmole/mg protein was found. In one patient with hyperplastic parathyroid glands secondary to renal failure, a high affinity EGF binding with Kd of 1.7 nM and Bmax of 18 fmole/mg protein was found. In one patient with persistent hyperparathyroidism following a successful renal transplant (tertiary hyperparathyroidism), a low affinity EGF binding with Kd of 25 nM and Bmax of 219 fmole/mg protein was found. The binding of EGF did not correlate with the preoperative serum calcium or PTH levels. Thus, hyperplastic parathyroid glands (either primary or secondary) have high affinity EGF receptors whereas solitary parathyroid adenomas do not.

Presence of neuron-specific enolase and somatostatin in human parathyroid tissues.

The association of parathyroid abnormalities with apudomas prompted us to examine parathyroid tissues for the presence of neuron-specific enolase and somatostatin. Enolase was present in extracts of 29 out of 29 parathyroid specimens; tissue content was significantly higher in adenoma than in hyperplasia tissues (p less than 0.005). Somatostatin was present in 14 of 33 specimens. Immunoreactive somatostatin measured in tissue extracts' fluids coeluted on Sephacryl chromatography along with synthetic somatostatin-14 in studies of two parathyroid carcinoma specimens. Since neuron-specific enolase has been found only in neural and neuroendocrine cells, our results suggest that human parathyroid glands may contain neuroendocrine elements. The differential content of neuron-specific enolase in adenoma versus hyperplasia specimens may be diagnostically useful in selected cases. The significance of the presence of somatostatin in some but not all parathyroid tumors requires further investigation. Taken together with our prior findings of gastrin and pancreatic polypeptide in some human parathyroid glands, we postulate that human parathyroid tumors contain neural crest elements.

Analysis of immunoreactive and biologically active human parathyroid hormone-peptides by high-performance-liquid-chromatography.

A combination of high-performance-liquid-chromatography (HPLC), sensitive radioimmunoassays and a homologous in vitro bioassay was used to characterise human parathyroid hormone (hPTH)-peptides in human parathyroid adenoma and plasma. Chromatography of several synthetic hPTH-peptides allows the calibration of the HPLC column. On the basis of sequence hydrophobicity the elution position of peptides can be predicted. A model for the determination of the minimal peptide sequence of each peptide has been developed which based on immunological and physico-chemical properties allows the characterisation of unknown hPTH-peptides. Using this technique the heterogeneity of circulating hPTH-peptides in human plasma has been examined. Plasma extracts from healthy individuals, osteoporotic, hyperparathyroid and pseudo-hyperparathyroid patients were investigated. A uniform pattern in the heterogeneity of hPTH-peptides was detected. Using parathyroid adenoma as reference disease specific changes were characterised.

Immunoreactive human chromogranin A in diverse polypeptide hormone producing human tumors and normal endocrine tissues.

Chromogranin A, the major soluble protein costored and coreleased by exocytosis with catecholamines from the adrenal medulla, has recently been detected in several bovine polypeptide hormone producing tissues. We therefore searched for chromogranin, by immunohistology, in human polypeptide hormone producing tumors as well as normal human endocrine tissues. The chromogranin A antigen was purified from catecholamine storage vesicles of human pheochromocytoma, to which rabbit antisera were developed, allowing immunohistologic studies by the indirect rabbit anti-peroxidase technique. Specific chromogranin staining was noted in all polypeptide hormone producing human tumors studied (pheochromocytoma chromaffin cells, n = 3; medullary thyroid carcinoma parafollicular C cells, n = 2; thyroidal C cell hyperplasia cells, n = 1; parathyroid adenoma chief cells, n = 1; pancreatic islet cell tumor islet cells, n = 1; oat cell carcinoma cell line M-103) as well as in all normal polypeptide hormone producing tissues (adrenal medulla chromaffin cells, parathyroid chief cells, thyroid parafollicular C cells, pancreatic islet cells, gut enteroendocrine cells, and anterior pituitary cells). Chromogranin may have a widespread distribution in human polypeptide hormone producing tissues, and may be a useful histologic marker for peptide producing tumors.

Presence of calmodulin in parathyroid adenomas.

A significant percent of parathyroid adenomas demonstrate decreased sensitivity to the suppressive effects of calcium on parathyroid hormone secretion. The recognition that calmodulin mediates a large number of calcium-dependent cellular events suggests that calmodulin may play a key role in the normal regulation by calcium of parathyroid activity. We therefore undertook to determine if parathyroid adenomas contained calmodulin and if so, if the calmodulin content of adenomas could be correlated with the serum concentrations of calcium and immunoreactive parathyroid hormone (IPTH) in hyperparathyroid patients prior to surgery. Calmodulin was assayed by activation of a calmodulin-stimulatable phosphodiesterase (PDE) obtained from rat brain. We found PDE-stimulating activity in each of six adenoma extracts examined. The stimulation had the following properties, which are characteristic of calmodulin-mediated processes: (1) it required the presence of calcium; (2) the dose response to adenoma extract and human red blood cell calmodulin were parallel, and (3) the stimulation was phenothiazine-inhibitable. The amount of calmodulin present in the adenoma extracts ranged from 0.4 to 1.25 units/micrograms protein. No correlation was found between the calmodulin content of the adenomas and the presurgical levels of either serum calcium or serum IPTH.