Parathyroid hormone in chronic renal failure: studies with two different parathyroid hormone radioimmunoassays.

Two assays for immunoreactive parathyroid hormone (iPTH) with different specificities were used to evaluate the role of iPTH measurement in patients with chronic renal failure (CRF). One measured largely C-terminal iPTH fragments, the other largely intact iPTH. In untreated CRF, the log iPRH for each assay was significantly correlated with the reciprocal of the creatinine clearance (CCr). C-terminal iPTH was elevated at relatively high CCr values, but intact iPTH was not elevated until later in the progression of CRF. In hemodialysis patients treated with 25-hydroxyvitamin D3, intct iPTH correlated better than C-terminal iPTH with clinical improvement. These two assays used together were more helpful in evaluation of CRF patients than either assay alone.

Radioimmunoassay for intact and carboxyl-terminal parathyroid hormone: clinical interpretation and diagnostic significance.

The phenomenal growth in our knowledge of parathyroid hormone (PTH) physiology, chemistry and radioimmunoassay in the past 15 years has produced a significant increase in the use of the assay in the clinical laboratory evaluation of patients with disorders of calcium homeostasis. Recent experience with assays that have specificities for different regions of the amino acid sequence of the hormone and that can thus measure different portions of the total immunoreactivity in blood suggests that there may be different clinical applications for such assays. This report describes two different radioimmunoassay procedures and the clinical experience with each and suggests how each assay may be utilized in clinical evaluation of possible parathyroid dysfunction. The assay for carboxyl-terminal PTH is more useful in the differential diagnosis of the possible causes of hypercalcemia, the intact PTH assay is preferred in selective venous catheterization for preoperative localization of hyperfunctioning tissues, and both assays have usefulness in the evaluation of patients with hypocalcemia. In chronic renal failure, the considerations are more complex. In many patients, the intact PTH assay is preferred for monitoring the clinical course; however, in other patients the carboxyl-terminal PTH assay has been more useful. The best assay for each patient must be determined by initial evaluation with both assays.

Carboxyl-terminal fragments of human parathyroid hormone in parathyroid tumors: unique new source of immunogens for the production of antisera potentially useful in the radioimmunoassay of parathyroid hormone in human serum.

We have found large quantities of immunoreactive carboxyl-terminal fragments of human parathyroid hormone )hPTH) in a previously discarded fraction [the 7.5% trichloroacetic acid (TCA)supernate] generated during extraction of intact hPTH from hyperfunctioning parathyroid tissue by the urea-TCA procedure. It is well established that serum RIAs directed toward the carboxyl-terminal region of hPTH are superior to those directed toward the amino-terminal region in the differential diagnosis of patients with suspected chronic parathyroid dysfunction. However, antisera that react with the carboxyl-terminal region of hPTH are not yet available for general use for these assays because of a lack of suitable hPTH immunogens. We immunized seven guinea pigs and two goats with the desalted 7.5% TCA supernate (containing about 2% carboxyl-terminal hPTH fragments); three of the guinea pigs and one goat produced high affinity antisera with predominant specificity for the carboxyl-terminal region of PTH. One of the guinea pig antisera had affinity for hPTH equal to that of our laboratory's best antiserum (GP1M) used in diagnostic RIAs for serum PTH. The use of this byproduct fraction as an immunogen should permit a large scale immunization program in large animals to provide standardized, species-and sequence-specific antisera potentially useful in RIAs for diagnosis of parathyroid disease.

Parathyrin radioimmunoassay: diagnostic utility of antisera produced against carboxyl-terminal fragments of the hormone from the human.

Antisera directed toward the carboxyl-terminal region of human parathyrin (parathyroid hormone), for use in daignostically applicable radioimmunoassays of the hormone in serum, are scarce, largely because of the lack of suitable immunogens of human origin. We produced four antisera in goats and guinea pigs by immunization with recently discovered carboxyl-terminal fragments of human parathyrin extracted from parathyroid tumors. Here, we report results of radioimmunoassays of nearly 200 normal and pathological sera with one of these antisera; we observed almost complete differentiation between concentrations of parathyrin in serum of healthy normal subjects and patients with primary, secondary (due to chronic renal failure), or "ectopic" hyperparathyroidism (due to nonparathyroid cancer). The availability of a new immunogen should now make possible the deliberate production of large quantities of diagnostically applicable parathyrin antisera directed toward the carboxyl-terminal region of human parathyrin. This should, in turn, lead to more widespread availability of this useful radioimmunoassay.

Immunoheterogeneity of parathyroid hormone in venous effluent serum from hyperfunctioning parathyroid glands.

The immunoreactive parathyroid hormone (iPTH) in the plasma of hyperparathyroid man consists largely of carboxyl (COOH)-terminal fragments of the hormone. Although these fragments have been thought to arise principally or solely from peripheral metabolism of intact human PTH {hPTH(1-84)} secreted from the parathyroid gland, there is disagreement about the source of iPTH fragments in vivo. To reexamine this question, we fractionated peripheral and thyroid or parathyroid venous effluent sera from four patients with primary hyperparathyroidism using a high-resolution gel filtration system (Bio-Gel P-150 columns run by reverse flow). The column effluents were analyzed using two PTH radioimmunoassays, one directed toward the amino(NH(2))-terminal region of the molecule, the other toward the COOH-terminal region. In all four thyroid or parathyroid venous effluent sera studied, iPTH was 9-180 times higher than in peripheral serum from the same patient; after fractionation, hPTH(1-84) accounted for only a portion of the total iPTH (35-55% with the assay directed toward the COOH-terminal region of hPTH, >90% with the NH(2)-terminal directed assay.) The remaining iPTH eluted from Bio-Gel P-150 after hPTH(1-84) as NH(2)-or COOH-terminal hPTH fragments. These results suggest that parathyroid tumors secrete large quantities of hPTH fragments. Based on estimates of their molar concentrations in serum, tumor-secreted COOH-terminal hPTH fragments could account for most of these peptides in peripheral serum if their survival times were, as estimated by several other workers, 5-10 times that of hPTH(1-84). We conclude that, in contrast to published information, secretory products of hyperfunctioning parathyroid tissue are probably a major source of serum PTH immunoheterogeneity.

Relative biologic activities of human and bovine parathyroid hormones and their synthetic, NH2-terminal (1-34) peptides, as evaluated in vitro with renal cortical adenylate cyclase obtained from three different species.

The relative biologic activities of native human parathyroid hormone, hPTH (1-84), native bovine parathyroid hormone, bPTH (1-84), and their respective synthetic, NH2-terminal, biologically-active (1-34) fragments were compared in vitro using adenylate cyclase preparations from human, chicken, and rat renal cortex. The concentrations of the hormones required for half-maximal stimulation of the enzymes were determined from dose response curves. bPTH (1-84) had greater apparent activity than hPTH (1-84), using rat or chicken renal adenylate cyclase, but, with human renal adenylate cyclase, the apparent activities of the two hormones were equal. Synthetic hPTH (1-34) possessed about 1/10 of the apparent activity of hPTH (1-84) in all three adenylate cyclase systems. However, (GLU22)bPTH (1-34) was about equal inapparent activity to bPTH (1-84) in the three systems. We propose that different rates of hormone degradation at or near renal receptor sites may be responsible for the dependence of the relative biologic activity on the assay system used. In the case of hPTH a peptide chain longer than (1-34) may be required for the full biologic activity of the hormone...

Parathyroid hormone receptors of renal cortex: specific binding of biologically active, 125I-labeled hormone and relationship to adenylate cyclase activation.

Biologically active (125)I-labeled bovine parathyroid hormone (prepared by electrolytic iodination) and its synthetic NH(2)-terminal (1-34) biologically active fragment bound rapidly and specifically to a purified plasma membrane preparation from bovine renal cortex. Binding of labeled intact hormone or labeled NH(2)-terminal (1-34) peptide was inhibited competitively by unlabeled (1-34) peptide in the same range of concentrations that activated renal cortical 3':5'-adenylate cyclase (EC 4.6.1.1) in these membranes. The concentrations of synthetic (1-34) peptide for half-maximal inhibition of binding of labeled hormone as well as half-maximal activation of the enzyme were about 0.6 muM (2.5 mug/ml). Therefore it is likely that the binding activity studied represents a physiologically important renal receptor for parathyroid hormone. Biologically inactive (oxidized) forms of parathyroid hormone and (1-34) NH(2)-terminal peptide as well as calcitonin, glucagon, insulin, and epinephrine failed to competitively inhibit the binding of labeled (1-34) parathyroid hormone or activate adenylate cyclase in the renal cortical membrane preparation. Observations with the NH(2)-terminal (1-34) biologically active fragment of parathyroid hormone suggest that the COOH-terminal region of the molecule is not required for receptor binding.