Joan M. Zanelli

A SENSITIVE BIOASSAY OF PARATHYROID HORMONE IN PLASMA

Understanding of calcium metabolism in health and disease has been retarded by the lack of an adequately sensitive bioassay of parathyroid hormone. The problem of dissociation of bioactivity and immunoactivity, well recognized for other polypeptide hormones, is exaggerated in the case of parathyroid hormone by the disproportionately long half‐time in the circulation of the immunoreactive fragments. A new method of assaying the biological activity of parathyroid hormone in plasma has been developed, based on the cytochemical methods which have yielded highly sensitive bioassays of other polypeptide hormones. It depends on the stimulation of glucose 6‐phosphate dehydrogenase activity in the distal convoluted tubules of segments of guinea‐pig kidney maintained in vitro, and measured by microdensitometry. The limit of sensitivity of the assay is 5 fg/ml (bPTH); the index of precision is 0.09 ± 0.04 (mean ± SEM; n= 11).

25 Years of salmon calcitonin: From synthesis to therapeutic use

ConclusionAlthough calcitonin has an established place in the treatment of Pagets disease of bone and certain disorders of calcium homeostasis, its most exciting clinical application is in the management of postmenopausal and senile osteoporosis. This disorder affects a vast number of women and, with the increasingly aging population, imposes a heavy burden on health care systems. Although other therapies for osteoporosis and other disorders of bone turnover (e.g., bisphosphonates, fluoride) are in use, many questions remain to be answered concerning their side effects and long-term safety, whereas salmon calcitonin is of proven safety. The introduction of salmon calcitonin nasal spray is a significant therapeutic advance, and the development of other noninjectable preparations will further increase the acceptability of calcitonin to patients and physicians alike.

Evidence that protease inhibitors reduce the degradation of parathyroid hormone and calcitonin injected subcutaneously.

1 Agents known to delay absorption from a subcutaneous site were tested in chicks for their ability to prolong the hypercalcaemic response to parathyroid hormone (PTH). 2 Polyvinylpyrrolidone was found to enhance the response but gelatine greatly reduced the 2 h hypercalcaemia. 3 The reduction by gelatine was reversed when the protease inhibitor aprotinin was added to the injection vehicle, and hypercalcaemia then persisted for more than 8 h. 4 Of other protease inhibitors studied, e‐aminocaproic acid was also found to enhance the hypercalcaemic response to subcutaneous PTH and its fragments but, unlike aprotinin, it was ineffective in the presence of gelatine. 5 By radioimmunoassay and bioassay respectively, it was confirmed that aprotinin raised circulating levels of PTH and also of another peptide hormone, calcitonin, injected subcutaneously. 6 Addition of calcium to the solutions injected subcutaneously abolished the hypercalcaemic response to PTH while injection of calcium and PTH simultaneously but at separate sites left the response unaltered. 7 The two protease inhibitors, e‐aminocaproic acid and aprotinin, each restored the response to subcutaneous PTH despite the presence of calcium at the injection site. 8 It was concluded that protease inhibitors injected subcutaneously with PTH and calcitonin in the chick reduced the rate of degradation of these hormones and that the proteases responsible for hormone degradation at the subcutaneous injection site may be released or activated by calcium ions.

Purification and assay of bovine parathyroid hormone by reversed-phase high performance liquid chromatography.

Reversed-phase high-performance liquid chromatography (HPLC) has been used to purify a crude extract of bovine parathyroid glands, in a single run on an analytical column, to give a high yield of homogeneous material with full bioactivity in in vivo bioassay. Bovine parathyroid hormone (bPTH) prepared and purified by conventional procedures has been rapidly and quantitatively separated from its oxidation and other degradation products, from hormone fragments and from non-hormonal contaminants. Recovery of bPTH, monitored by region-specific immunoassays, in vivo bioassay and re-chromatography on HPLC was greater than 93%. The detection limit of the HPLC system, using endogenous tryptophan fluorescence, was 20 ng bPTH.

An Homologous and Sensitive Radioimmunoassay for the Synthetic Amino-Terminal (1-34) Fragment of Human Parathyroid Hormone: Application to the Clearance of This Peptide Administered in Vivo

The synthetic 1-34 amino-terminal fragment of human parathyroid hormone (hPTH 1-34) is undergoing multicentre clinical trials to assess its long term therapeutic potential in the treatment of osteoporosis. An homologous radioimmunoassay (reagents prepared from the synthetic hPTH 1-34 peptide) has been developed to monitor the pharmacokinetics of hPTH 1-34 in man and in a dog model. The assay is rugged, sensitive (detection limit 1.75 x 10(-11) moles/litre) and precise (coefficient of variation 6%). Three different ampouled preparations of the native intact hPTH 1-84, of different degrees of purity (approximately 3%-90% pure) gave complete log dose response curves parallel to that of the ampouled synthetic hPTH 1-34 peptide, and were equipotent on a molar basis. Native intact bovine PTH 1-84 showed an incomplete non-parallel displacement curve; there was no recognition of synthetic hPTH 44-68 and 53-84 peptides. Preliminary application of the assay to the determination of the plasma disappearance of hPTH 1-34 in man and dog gave half-times (t1/2) of 3-8 minutes for a first exponential component and 12-18 minutes for the second; in the dog, metabolic clearance rate was calculated to be 9ml/kg/minute and the distribution space 160ml/kg.

High-performance liquid chromatographic methods for the analysis of human parathyroid hormone in reference standards, parathyroid tissue and biological fluids

Reversed-phase high-performance liquid chromatography (RP-HPLC) has been used to fractionate human parathyroid hormone (hPTH) from a variety of natural sources and to compare it with synthetic hPTH and hPTH fragments. Multiple radioimmunoassay systems for amino, mid and carboxyl regions of hPTH were used to monitor various preparations of hPTH previously prepared by conventional methods and ampouled in nanogram amounts for reference standard and reagent purposes. Results confirmed that they were free of detectable cleavage products, but showed that the intact hPTH comprised three or four closely associated components. A similar pattern of heterogeneity was obtained when hPTH was extracted from stored human parathyroid adenomata by a simple rapid HPLC bulk fractionation method. Comparison with synthetic 1-84 hPTH and modification of sample handling to minimize oxidative conditions, indicate that some of these components are probably intermediate oxidation products. A number of less hydrophobic components, with carboxyl region immunoreactivities, were obtained from the individual adenoma samples, human parathyroid cyst fluid, ampouled samples of human adenoma tissue culture medium, and secondary hyperparathyroid plasma ultrafiltrate when they were fractionated by RP-HPLC. The results strongly suggest that the biological degradation of hPTH is more complex than generally believed, and that RP-HPLC offers a new dimension in its analysis.

Approaches to the Study of the Function and Activity of Bone Regulatory Factors: Established and Potential Methods

The aim of this paper is to review methodological approaches which can either be used to explore the biological activities of bone regulatory factors or be used to measure the amount of a particular factor. The discrimination between explore and measure needs to be made at the outset because practical and scientific considerations for each of these aspects can, at times, appear to be mutually exclusive. This is not intended to be a comprehensive review of methodologies and we do not propose to compare and contrast individual methods for individual substances; instead, we aim to review the rationale of choosing, or developing, appropriate laboratory techniques. We will also propose potential methods, based on in situ biochemistry, which are expected to make a major research contribution to the study of hone regulatory factors. Selection of the most appropriate methodologies requires an objective assessment of the scientific questions to be asked and the relevance of the experimental answers likely to be obtained.

Large Scale Screening Programme for Selection of Antisera for Radioimmunoassay of Human Parathyroid Hormone

A large-scale, three-phase screening programme has been devised for the rapid selection of antisera which might be of potential use in clinical radioimmunoassays for human parathyroid hormone. A total of 122 sera from 169 guinea pigs and 6 rabbits immunized with bovine parathyroid hormone, and 12 guinea pigs immunized with human parathyroid hormone were assessed relative to reference antisera. Pre-determined criteria for the three phase programme were imposed by the requirement for antisera that could be used at dilutions greater than 10(-5) in order to ensure continuity of supplies for wide-spread distribution and by the limited availability of human parathyroid hormone for testing purposes. Of the sera tested, only 5 were selected as having high titre and sensitivity for low concentrations of human parathyroid hormone. The 5 antisera were further evaluated for amino- and carboxyl-region specificities for the human parathyroid hormone in comparison with antiserum 211/32, widely distributed for use in radioimmunoassay for clinical purposes. The selected antisera appear to be of high affinity with good recognition of the whole or carboxyl-region parts of the human parathyroid molecule.

International Standards for salmon calcitonin, eel calcitonin, and the Asu1-7 analogue of eel calcitonin: calibration by international collaborative study.

Regulatory specifications in most countries require that the potency of salmon calcitonin (sCT) clinical products be expressed in International Units (IU) defined by the World Health Organization (WHO) International Standard. The first ampouled standard was prepared in 1972 and has been distributed world-wide since then. A batch of ampoules to serve as the replacement standard is now required. Other piscine calcitonins, eel calcitonin (eCT) and an amino-suberic acid analogue of eCT (Asu1-7 eCT) are now clinical products in some countries and international standards are required for these peptides which are similar to, but not identical with, sCT. This paper describes the preparation of three new ampouled standards and their biological calibration by international collaborative study comprising 17 participants from 10 countries. Following the recommendations in the final report of the collaborative study, the 2nd International Standard (IS) for sCT, the 1st IS for eCT and the 1st IS for Asu1-7 eCT were recently established by WHO, each with an assigned potency in IU, and are now available for issue.

Quantitative cytochemical responses to exogenously administered calcitonins in rat kidney and bone cells

Time- and dose-dependent changes in intracellular enzyme activities in kidney and bone from rats injected with calcitonin have been assessed by quantitative cytochemistry. The doses of salmon calcitonin given were similar to those suggested in the Pharmacopoeial rat hypocalcaemia bioassay (1-50 mIU/50 g body weight). The highest doses produced 30% inhibition of alkaline phosphatase activity, maximal within 20 min after injection, in cells of renal proximal tubules and a stimulation of calcium-dependent adenosine triphosphatase activity in kidney cortical and outer medullary cells. Alkaline phosphatase activity in the periosteal bone cells was markedly inhibited at the lowest doses. When doses of human and porcine calcitonins were given which would be equipotent with that of salmon calcitonin in the rat hypocalcaemia bioassay, the effect of the non-mammalian peptide on renal alkaline phosphatase activity was relatively greater than that of the mammalian peptides. Oxidized human calcitonin did not inhibit renal alkaline phosphatase activity even when an amount equivalent to 10 times the highest dose of the unmodified peptide was injected.