Circulating levels of biologically active and immunoreactive intact parathyroid hormone in human newborns.

We evaluated circulating levels of biologically active and immunoreactive intact parathyroid hormone [iPTH-(1-84)] in 47 newborns at birth and eight hypocalcemic preterm infants during the first 10 d of life. Use of two sensitive detection systems, the cytochemical bioassay and an immunoradiometric assay specific for intact parathyroid hormone, enabled us to compare plasma concentrations of PTH-like bioactivity (bioPTH) and iPTH-(1-84). Mean umbilical venous plasma bioPTH was elevated in nondiabetic term and preterm newborns [22.5 +/- 3.1 (+/- SEM) and 15.8 +/- 2.5 ng-equiv/L, respectively] compared with normal adult subjects (9.8 +/- 2.6 ng-equiv/L; p less than 0.01). Umbilical bioPTH was suppressed in five term infants of diabetic mothers (2.6 +/- 0.4 ng-equiv/L). In contrast, iPTH-(1-84) was low in term and preterm nondiabetic infants' and term infants of diabetic mothers' umbilical samples (5.4 +/- 1.5, 4.3 +/- 1.5, and 2.4 +/- 1.0 ng/L, respectively). Umbilical venous bioPTH was highly correlated with the magnitude of the transplacental calcium gradient (r = 0.90; p less than 0.05). In eight preterm infants studied longitudinally, by 24-36 h of life, declining plasma total and ionized calcium (1.71 +/- 0.04 and 0.78 +/- 0.03 mmol/L, respectively) were accompanied by a significant rise in both bioPTH (41.2 +/- 6.3 ng-equiv/L) and iPTH-(1-84) (56.3 +/- 11.6 ng/L). These data indicate that the 3rd trimester fetoplacental circulation contains levels of bioPTH several-fold higher than those of immunoreactive intact hormone. We also conclude that even hypocalcemic preterm newborn infants can significantly elevate circulating levels of PTH.

A multicenter, randomized, double-blind trial of somatostatin in the management of acute hemorrhage from esophageal varices.

A prospective, randomized, placebo-controlled, double-blind, multicenter clinical trial of intravenous somatostatin (Stilamin; Serono Laboratories, Inc., Randolph, MA) was performed in 102 patients with actively bleeding esophageal varices from August, 1985, to November, 1986. Patients had major hemorrhage indicated by hematemesis or melena and evidence of significant blood loss. For entry, patients had to have endoscopic demonstration of active bleeding from esophageal varices or stigmata of recent hemorrhage and bright red blood in the gastric aspirate with no other source of bleeding found. Randomized patients received identical-appearing somatostatin or placebo for a 30-hr study period. Those given somatostatin received a 250-micrograms bolus and a 250-micrograms per hr infusion with repeat bolus and doubling of the infusion if the bleeding was not controlled. In retrospect, 18 patients could not be evaluated. Of the 84 evaluable patients, 48 received somatostatin and 36 placebo. They were comparable in age, gender, severity of liver disease and history of variceal bleeding. Transfusion requirements were similar in both groups. Bleeding stopped for 12 consecutive hr during 30 hr of the study period in 31 (65%) of the somatostatin group vs. 30 (83%) of the placebo group (p = 0.06). The median time to cessation of bleeding was 2 hr in the placebo group and 3 hr in the somatostatin group. Deaths following the study period were nine (25%) in the placebo group and 15 (31%) in the somatostatin group. Within the limitations of the present study, we conclude that somatostatin was ineffective in the management of active bleeding of esophageal varices.

Microinjected Xenopus oocytes secrete mature, biologically active parathyroid hormone.

Xenopus oocytes have been shown to faithfully translate, process, and secrete a number of secretory proteins after the injection of heterologous mRNAs. The oocyte has the capacity to perform a variety of posttranslational protein modifications but has been reported to be incapable of carrying out certain two-step cleavages which proceed via propeptide intermediates. We examined the ability of the oocyte to process preproPTH after the injection of parathyroid mRNA. Microinjected oocytes secreted material which could be detected in a sensitive cytochemical bioassay for PTH. This activity paralleled that of the PTH standard in the assay and was entirely eliminated by a competitive inhibitor of PTH binding, by preincubation with an anti-PTH antiserum, and by coinjecting oocytes with an oligonucleotide mixture complementary to PTH sequences. Immunoprecipitable proPTH and PTH were present in oocyte homogenates, but oocyte-conditioned medium contained only mature PTH(1-84). We conclude that the Xenopus oocyte is capable of accurately processing preproPTH to the mature secretory form of the peptide.

The calcium channel blocker diltiazem lowers serum parathyroid hormone levels in vivo and in vitro.

PTH secretion is inversely related to the extracellular and cytosolic calcium (Ca2+) concentrations and, therefore, might be affected by calcium channel blockers such as diltiazem. To investigate the effects of diltiazem on parathyroid function in vivo, 15 subjects were treated with diltiazem (120-360 mg/day), and 15 subjects were treated with the nonspecific vasodilator hydralazine (75-150 mg/day). Diltiazem lowered serum PTH levels from 1.07 +/- 0.07 to 0.87 +/- 0.07 pg/L (P = 0.001), and increased urinary calcium and decreased urinary phosphate excretion (P less than 0.001 and P less than 0.01, respectively). The hydralazine-treated subjects had no significant differences in any of these parameters. To investigate this effect further, dispersed bovine parathyroid cells were incubated for 2 h with or without diltiazem. Regression analysis of PTH released vs. the concentration of diltiazem (10(-7)-10(-4) mol/L) revealed a significant negative relationship (P less than 0.01) with 40% inhibition of PTH release at 10(-4) mol/L (P less than 0.01). The cytosolic Ca2+ concentration, measured using the Ca2+-sensitive fluorescent dye fura-2, was significantly increased in the presence of 10(-4) mol/L diltiazem (P less than 0.01). In summary, diltiazem lowered PTH levels in vivo and in vitro, perhaps acting as a Ca2+ channel agonist in the parathyroid cell and inhibiting PTH release through a rise in the cytosolic Ca2+ concentration.

Effects of estrogen on mineral metabolism in postmenopausal women as evaluated by multiple assays measuring parathyrin bioactivity.

Data on the effect of estrogen on immunoreactive parathyrin (iPTH) in postmenopausal women are conflicting. We administered estrogen or placebo to 21 postmenopausal women for 12 weeks and measured PTH bioactivity (bioPTH), using the renal cytochemical bioassay. Before treatment, there was a negative correlation between nephrogenous cAMP and the tubular maximum for urinary phosphate excretion and a positive correlation between values measured by a mid-region-specific PTH RIA and those measured in an immunoradiometric assay for intact PTH. Values measured by the midregion-specific RIA were also positively correlated with nephrogenous cAMP. BioPTH values were not correlated with other indices of PTH activity but were increased compared with values for younger subjects. After estrogen treatment there was no change in bioPTH activity despite an early decrease in serum osteocalcin and a later increase in nephrogenous cAMP. PTH concentrations measured by mid-region-specific or intact RIAs were unchanged, but sample size may have been insufficient to exclude the possibility of significant changes in these values. The effects of estrogen on mineral metabolism in postmenopausal women are time-dependent. Early effects are independent of PTH, and later effects are variably associated with increased PTH activity.

Serum parathyroid hormone (PTH) in pregnant women determined by an immunoradiometric assay for intact PTH.

Most studies of circulating PTH levels using traditional RIAs have supported the concept of physiological hyperparathyroidism of pregnancy, with pregnant women having serum immunoreactive PTH levels significantly higher than those in nonpregnant subjects. However, such RIAs are insensitive and often detect inactive PTH fragments, so that the correlation between PTH immunoreactivity and bioactivity is poor. Employing a new intact PTH immunoradiometric assay (Allegro-Nichols), we reassessed the effects of pregnancy on parathyroid function. The mean serum PTH level in 81 pregnant women was 14.4 +/- 6.3 (+/- SD) compared to 24.8 +/- 9.0 ng/L in 11 normally cycling nonpregnant women (P less than 0.001). The mean serum total and ionized calcium levels in the 2 groups were similar. In 5 of the pregnant women, serum bioactive PTH, determined by cytochemical bioassay, was slightly lower (7.7 +/- 3.4 ng/L) than in normal individuals (11.1 +/- 1.9 ng/L). Our findings suggest, in contrast with the results of most previous studies, that serum intact PTH may decline during pregnancy.

Identification of transcripts encoding a parathyroid hormone-like peptide in messenger RNAs from a variety of human and animal tumors associated with humoral hypercalcemia of malignancy.

The syndrome of humoral hypercalcemia of malignancy (HHM) appears to be mediated in many instances by a parathyroid hormone-like peptide, which has recently been purified, sequenced, and cloned. Using a probe representing the coding region of the human PTH-like peptide, we examined by Northern analysis poly (A)+ RNA from a variety of human and animal tumors associated with HHM. Hybridizing transcripts were identified in mRNA from each of 12 human and each of four animal HHM-associated tumors, with a complex hybridization pattern observed in the human mRNAs and a relatively simple pattern observed in the animal mRNAs. Poly (A)+ RNA prepared from tumors of similar histological types unassociated with HHM failed to hybridize with the probe. Messenger RNA-dependent biological activity from the animal tumors was entirely eliminated in a hybridization-arrest experiment using a complementary oligonucleotide spanning the region of homology between human PTH and the PTH-like peptide. These findings indicate that the PTH-like peptide is associated with the syndrome of HHM in a wide spectrum of tumor types from a variety of mammalian species and that the PTH-like sequence in the proximal amino terminus of the peptide is highly conserved.

A novel neuroendocrine cell surface glycoprotein: identification, isolation, and initial characterization.

Murine monoclonal antibodies (MAbs) HISL-5, -9, and -14, generated after immunization of mice with human pancreatic islet cell preparations, recognize a differentiation antigen expressed by the pancreatic islet cells. These MAbs react strongly with all endocrine cell subtypes of human pancreatic islets, but minimally if at all with the exocrine acinar cells, vascular cells, and stromal connective tissue cells of the pancreas. The antigen is located on the cell surface (plasma membranes), as indicated by immunofluorescence staining of viable cell preparations. Besides the pancreatic islets, HISL-5, -9, and -14 antigenic determinants are also expressed by thyroid follicular cells, parathyroid chief cells, and anterior pituitary cells, other commonly involved targets in organ-specific autoimmune disorders. Preliminary biochemical findings indicated that the MAb-defined epitope(s) is trypsin sensitive and resistant to periodate oxidation and exposure to chloroform-methanol. Further biochemical studies, including single step MAb immunoaffinity chromatographic purification, indicate that the antigen recognized by the MAbs HISL-5, -9, and -14 is a 100 K glycoprotein.

Identification of a cDNA encoding a parathyroid hormone-like peptide from a human tumor associated with humoral hypercalcemia of malignancy.

Humoral hypercalcemia of malignancy is a common paraneoplastic syndrome that appears to be mediated in many instances by a parathyroid hormone-like peptide. Poly(A)+ RNA from a human renal carcinoma associated with this syndrome was enriched by preparative electrophoresis and used to construct an enriched cDNA library in phage lambda gt10. The library was screened with a codon-preference oligonucleotide synthesized on the basis of a partial N-terminal amino acid sequence from a human tumor-derived peptide, and a 2.0-kilobase cDNA was identified. The cDNA encodes a 177 amino acid protein consisting of a 36 amino acid leader sequence and a 141 amino acid mature peptide. The first 13 amino acids of the deduced sequence of the mature peptide display strong homology to human PTH, with complete divergence thereafter. RNA blot-hybridization analysis revealed multiple transcripts in mRNA from tumors associated with the humoral syndrome and also in mRNA from normal human keratinocytes. Southern blot analysis of genomic DNA from humans and rodents revealed a simple pattern compatible with a single-copy gene. The gene has been mapped to chromosome 12.

Effects of extracellular calcium and magnesium on cytosolic calcium concentration in Fura-2-loaded bovine parathyroid cells.

A newly developed calcium-sensitive dye, Fura-2, was employed in dispersed bovine parathyroid cells to study the effects of extracellular calcium and magnesium on cytosolic calcium concentration and parathyroid hormone (PTH) release. In comparison with control cells, Fura-2-loaded parathyroid cells showed the same maximal rate of PTH release, set-point for extracellular Ca++ (the calcium concentration producing half of the maximal inhibition of PTH release), and maximal inhibition of PTH release (71.6%) by high extracellular Ca++. At an extracellular Mg++ concentration of 0.5 mM, raising extracellular Ca++ in a stepwise fashion from 0.5 mM to 2.0 mM produced a dose-dependent, statistically significant (p less than 0.01) increase in cytosolic Ca++ from 198 +/- 24 nM (0.5 mM Ca++) to 411 +/- 21 nM (2.0 mM Ca++) which closely paralleled the concomitant decrease in PTH release. An elevation of extracellular Mg++ from 0.5 mM to 5 mM, at an extracellular Ca++ of 0.5 mM, resulted in a transient spike of cytosolic Ca++ which lasted for approximately 30 seconds, followed by a small but stable increase in the cytosolic Ca++ concentration (174 +/- 7 nM vs. 237 +/- 10 nM, n = 4, p less than 0.01). Prior removal of extracellular calcium by addition of an excess of EGTA did not abolish the transient spike induced by high extracellular magnesium concentrations in Fura-2-loaded cells, suggesting that this rapid increase in cytosolic Ca++ arises, at least in part, from intracellular stores of Ca++. This is supported by the observation that pretreating cells with ionomycin resulted in disappearance of the magnesium-induced spike.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibodies to a human islet cell surface glycoprotein: 4F2 and LC7-2.

Monoclonal antibodies 4F2 and LC7-2 react with a cell surface differentiation antigen expressed by the endocrine cells of the human pancreatic islet, but not by the acinar pancreatic, ductular, vascular, or stromal connective tissue cells. Western immunoblotting procedures demonstrate the reactivity of the monoclonal antibody 4F2 with a 120 kilodalton islet cell protein in detergent-solubilized cell extracts. These two monoclonal antibodies have potential for application in many aspects of islet cell research and diabetes in general.

Monoclonal antibody-mediated modulation of parathyroid hormone secretion by dispersed parathyroid cells.

Available data suggest that ionized calcium may interact with a cell surface "sensor" or "receptor" to produce changes in one or more intracellular second messengers that ultimately regulate the release of parathyroid hormone (PTH). Recently, we developed a series of monoclonal antibodies directed toward specialized differentiation antigens expressed on endocrine cells. Since many of these monoclonal antibodies displayed exquisite specificity for cell surface molecules on the parathyroid cell, we used these reagents as probes to investigate signal recognition/transduction mechanisms associated with abnormal calcium-regulated PTH secretion. Depending on their binding site on the respective target antigen molecules, these monoclonal antibodies either stimulated or inhibited hormone secretion. Thus, defects in membrane-associated structures may contribute to deranged calcium-regulated PTH secretion in abnormal parathyroid cells.

Binding of monoclonal antibody (4F2) to its cell surface antigen on dispersed adenomatous parathyroid cells raises cytosolic calcium and inhibits parathyroid hormone secretion.

In the course of characterizing monoclonal antibodies (MAbs) recognizing cell surface antigens on dispersed human parathyroid cells (dPTCs), we identified one MAb (4F2) that bound avidly to parathyroid cells and had marked effects on parathyroid function. The binding of MAb 4F2 to human adenomatous dPTCs resulted in a marked [53.8 +/- 7.9% (+/- SEM)] reduction in low calcium (Ca)-stimulated PTH secretion to levels equivalent to those in cell suppressed by high extracellular Ca (1.5 mM). Typically, these functional effects were optimal at antibody dilutions of 1:10(4) to 1:10(5). Cell viability was confirmed at the conclusion of each experiment by trypan blue exclusion (greater than 90-95%) and cell surface immunofluorescence. Parallel studies using the Ca-sensitive dye Quin-2 showed that inhibition of PTH secretion in 4F2-treated cells was associated with a concomitant increase in cytosolic Ca (Cai) of 188% in 0.5 mM Ca; these values also approached Cai levels in control cells incubated in high Ca. Mab controls, P3 X 63, which do not bind to dPTCs, and Mab LC7-2, which recognizes a different epitope of the same antigen as 4F2 on dPTCs, did not alter PTH secretion or Cai. Immunoprecipitation of 125I-labeled parathyroid cell extracts with MAb 4F2 demonstrated proteins with mol wt of approximately 145, 85, and 45 under nonreducing conditions and 85 and 45 kilodaltons after reduction with 5% mercaptoethanol. These studies suggest that 1) Mab-4F2 binding to its cell surface antigen inhibits PTH secretion by human adenomatous parathyroid cells in vitro; 2) the alterations in secretory function could be related to by an attendant increase in Cai; 3) the 4F2 antigen on dPTCs is a heterodimeric protein of (approximately) 85K and 45K; and 4) the 4F2 antigen may be an important component of the Ca-sensing and/or signal-transducing mechanism in this cell.

Ouabain and low extracellular potassium inhibit PTH secretion from bovine parathyroid cells by a mechanism that does not involve increases in the cytosolic calcium concentration.

We have previously found that high extracellular calcium (Ca++) concentrations inhibit PTH release in association with a threefold to fourfold rise in cytosolic Ca++ concentration. Recent data have also shown that low extracellular potassium (K+) concentration or ouabain also inhibits PTH release to an extent comparable to that seen with high Ca++ and produce a marked rise in the intracellular sodium (Na+) content. These results suggested that low K+ and ouabain might modulate PTH release through increases in cytosolic Ca++ related to alterations in Na+-Ca++-exchange. In the present studies, we have examined further the mechanism(s) by which inhibition of the Na+-K+-ATPase regulates PTH release. Exposure of cells loaded with the Ca++-sensitive dye QUIN-2 to low K+ produced a 10% to 17% increase in cytosolic Ca++ at 0.5 to 1.0 mmol/L extracellular Ca++, which was statistically significant only at 0.75 mmol/L Ca++. In contrast, low K+ caused a statistically significant decrease in cytosolic Ca++ at 1.5 to 2 mmol/L Ca++, while ouabain lowered cytosolic Ca++ significantly by 23% to 46% at all Ca++ concentrations examined (0.5 to 2 mmol/L). Low K+ or ouabain had no effect on cellular levels of ATP or GTP or intracellular pH measured using the pH-sensitive dye BCECF [2', 7'-bis(carboxyethyl)-5,6-carboxyfluorescein]. The inhibition of secretion by low K+ or ouabain, unlike that due to high extracellular Ca++, was not reversed by TPA (12-O-tetradecanoyl phorbol 13-acetate), an activator of protein kinase C. Low K+ did produce a modest (30% to 40%) lowering of agonist-stimulated but not basal cAMP content.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid cell surface autoantibodies that inhibit parathyroid hormone secretion from dispersed human parathyroid cells.

Serum autoantibodies directed toward antigenic determinants on the surface of human parathyroid cells (PTAb-CS) have been demonstrated in a subset (8 of 23) of adult patients with idiopathic hypoparathyroidism (IHP). In sera from 3 of 8 patients with PTAb-CS, binding of these autoantibodies to their respective parathyroid cell surface antigen(s) resulted in marked inhibition of parathyroid hormone (PTH) secretion in an in vitro dispersed human parathyroid cell (dPTC) system. In 1 subject evaluated longitudinally, circulating levels of PTAb-CS, and the magnitude of the inhibitory effect on PTH secretion, temporally correlated with the clinical course of the hypoparathyroidism. These findings suggest a causative role for antibodies directed against cell surface antigens in parathyroid dysfunction in some cases of "autoimmune" hypoparathyroidism.

Cytochemical bioassay of circulating concentrations of rat parathyroid hormone: application to a study of age and sex.

We have found that the cytochemical bioassay (CBA) method, originally developed to measure circulating levels of biologically active parathyroid hormone (bioPTH) in humans, also can measure endogenous concentrations of PTH in rats. Therefore, we have applied this assay method to examine the relationships between age and sex and the circulating levels of bioPTH, calcium, and phosphorus in Fischer rats. The concentration of bioPTH increased in both males and females from 5 to 15 months of age (p less than .001) with no significant sex-related difference. Mean bioPTH values ranged from 2.3 to 3.6 pg (human PTH equivalents)/ml; these values were much lower (1 to 2 orders-of-magnitude) than those reported by others who used radioimmunoassays for PTH that do not discriminate between biologically active and inert PTH fragments. The CBA values for bioPTH were also 1/4-1/5 those obtained in rat serum by newer, more sensitive methods, the N-terminal specific PTH radioimmunoassay and the bioassay based on stimulation of chick renal adenylate cyclase. Regression analysis did not reveal any significant correlation between serum calcium and age. However, serum inorganic phosphorus decreased significantly (p less than .001) from 5 to 15 months of age in both male and female rats and was much lower in females than in males (p less than .001). The fall in serum phosphorus was negatively correlated with the rise in bioPTH (p less than .001). This study, the first to our knowledge to use a CBA to detect physiological changes in the circulating levels of bioPTH in rats, demonstrates the usefulness of the assay in experiments in this species.

Primary hyperparathyroidism with low serum 1,25-dihydroxyvitamin D levels.

Primary hyperparathyroidism is usually associated with normal or elevated serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] levels. We report a patient with extreme hypercalcemia (serum calcium, 19.4 mg/dl), primary hyperparathyroidism, and a very low plasma concentration of 1,25-(OH)2D. Surgical removal of a large parathyroid adenoma was associated with a decrease in the serum calcium and immuno- and bioactive PTH concentrations and normalization of the 1,25-(OH)2D level. The postoperative course was complicated by severe protracted hypocalcemia and cardiac arrest, requiring treatment with large doses of calcium iv. The low concentrations of 1,25-(OH)2D in this patient are an unusual manifestation of primary hyperparathyroidism, probably due to suppression of renal 1 alpha-hydroxylase activity by the severe hypercalcemia. We conclude that in severe hypercalcemia, a low serum 1,25-(OH)2D level does not exclude the diagnosis of primary hyperparathyroidism.

Abnormal parathyroid function in the X-linked hypophosphatemic mouse.

Employing a cytochemical bioassay, we compared parathyroid function in normal and X-linked hypophosphatemic (Hyp) mice. Under basal conditions Hyp mice manifested hypocalcemia and, in accord, had a plasma bioactive parathyroid hormone concentration (3.04 +/- 0.14 pg/ml) significantly greater than that of normals (2.16 +/- 0.14 pg/ml). We confirmed the validity of the bioassay by demonstrating that the plasma collected from both mouse models diluted parallel to the assay standard curve. Moreover, after parathyroidectomy, normal and Hyp mice had plasma bioactive parathyroid hormone levels approximately 90% less than those obtained under basal conditions and indistinguishable from one another. In further studies we observed that dietary calcium and/or vitamin D deprivation in both animal models resulted in a comparable decline of the plasma calcium concentration. However, the concordant increase of the circulating bioactive parathyroid hormone level was greater in the normal mice. Thus, the bioactive parathyroid hormone concentration obtained in response to a low calcium challenge in normals was significantly greater than that in Hyp mice. In contrast, in response to dietary calcium loading, the plasma bioactive parathyroid hormone levels did not decrease significantly from basal values in either animal model. These data illustrate that the bioactive parathyroid hormone concentration in both normal and Hyp mice is inversely correlated with the plasma calcium. However, while the Hyp mice maintain an elevated plasma parathyroid hormone concentration under basal conditions (in response to a decreased plasma calcium), the parathyroid activity of the mutants after a more severe hypocalcemic challenge is attenuated, resulting in a significantly different model of linear correlation. Thus, these data indicate that Hyp mice manifested abnormal regulation of parathyroid function.