Parathyroid Hormone (1-84): Assay Development and Application to the Physiology and Pathophysiology of Calcium Homeostasis

1. The initial aim of the thesis was the production of monoclonal antibodies for use in the development of an assay for PTH (1-84). The response to immunisation, using chemically synthesised PTH peptides as immunogen, was species and strain specific with DA rats consistently responding with high titre antibodies to PTH (1-34). Five monoclonal antibodies were produced to PTH (1-34), three of which (3B3, 4G3 and 6E3) were considered to be of potential use in assay systems. 2. A two-site immunometric assay specific for PTH (1-84) has been developed using 3B3 and a C-terminal monoclonal antibody - ESQ1. The assay development studies revealed 3B3 to be sensitive to the oxidation state of PTH. Hydrogen peroxide is therefore included in the assay to convert endogenous PTH (1-84) to the oxidised form prior to measurement. The validation studies confirmed the assay to be unaffected by PTH fragments. The assay has a minimum detection limit of 0.5 pmol/1, sufficient to measure PTH (1-84) in all normal subjects, and a range of 1. 5-250 pmol/1 with an intra-assay CV of less than 10% (2.8-250 pmol/1 less than 5% CV). Studies on clinical samples indicate good discrimination between normal subjects (mean 2. 1; range 1.0-5.0 pmol/1) and patients with primary hyperparathyroidism (mean 21.; range 5.8-100 pmol/1) who in turn are well separated from patients with hypercalcaemia of malignancy (14/180.5 pmol/1). 3. The measurement of PTH(1-84) over a 24 h period has shown the existence of circadian rhythm, characterised by an early evening rise and a broad peak through the night, in normal subjects which is absent in patients with primary hyperparathyroidism. The physiological significance of this observation is reflected in the presence of parallel changes in nephrogenous cyclic adenosine monophosphate in the normal subjects which are also absent in the hyperparathyroid patients. Although transient falls in calcium were observed these studies the changes in PTH (1-84) over the 24 h period could not be fully accounted for by changes in serum adjusted calcium concentrations. An important observation from these studies was that discrimination between normal subjects and patients with primary hyperparathyroidism is time dependent. Thus for optimal discrimination between normal subjects and patients with primary hyperparathyroidism PTH (1-84) samples should be taken, and reference ranges established, between 1000 and 1600 h. The most significant finding of the circadian studies was that PTH (1-84) concentrations in normal subjects over a 24 h period show a stronger temporal correlation with other circadian hormones, notably prolactin, than with adjusted calcium concentrations. This observation suggests that factors other than plasma ionised calcium are involved in the tonic control of PTH (1-84) secretion. 4. PTH (1-84) concentrations in the circulation vary in a manner consistent with pulsatile secretion. The estimates of pulsatility obtained in normal subjects, pulses every 10-15 minutes, are in agreement with the frequency of pulsatility previously reported for bioactive PTH. This supports the concept of PTH (1-84) being the major bioactive form of PTH in the circulation. The response to an acute lowering of serum calcium confirms that, in common with other hormones, modulations of the amplitude and frequency of pulses are an integral part of the control of PTH (1-84) secretion. However, the pattern of pulsatility observed, at the frequencies studied, appears to be characteristic of the individual and does not correlate with the presence of primary hyperparathyroidism. 5. The response to treatment, with the bisphosphonate Pamidronate, was compared in patients with either Paget's disease of bone or hypercalcaemia associated with malignancy. In the patients with Paget's disease therapy with Pamidronate produced a small but significant drop in serum adjusted calcium which was sustained throughout the study. This was accompanied by a significant rise in PTH (1-84). The concentration of PTH (1-84) continued to rise progressively despite no further fall in serum calcium. In patients with hypercalcaemia of malignancy Pamidronate produced the expected and prolonged decrease in serum adjusted calcium such that at the end of the study the mean calcium in these patients was within the reference interval. The PTH (1-84) concentrations rose progressively throughout the study to a mean concentration above the reference interval. More interestingly the initial increase occurred when the mean adjusted calcium was above the reference interval with no patient hypocalcaemic. The results suggest a resetting of the 'trigger' point for PTH (1-84) secretion in patients with hypercalcaemia of malignancy. An important conclusion from this study is that in the assessment of PTH (1-84) concentrations in the hypercalcaemic patient samples should be obtained prior to treatment being initiated