Structure of non-(1-84) PTH fragments secreted by parathyroid glands in primary and secondary hyperparathyroidism

Background. Non-(1-84) parathyroid hormone (PTH) fragments are large circulating carboxyl-terminal (C) fragments with a partially preserved amino-terminal (N) structure. hPTH (7-84), a synthetic surrogate, has been demonstrated to exert biologic effects in vivo and in vitro which are opposite to those of hPTH (1-34) on the PTH/PTHrP type I receptor through a C-PTH receptor. We wanted to determine the N structure of non-(1-84) PTH fragments. Methods. Parathyroid cells isolated from glands obtained at surgery from three patients with primary hyperparathyroidism and three patients with secondary hyperparathyroidism were incubated with S-35-methionine to internally label their secretion products. Incubations were performed for 8 hours at the patient-ionized calcium concentration and in the presence of various protease inhibitors. The supernatant was fractionated by high-performance liquid chromatography (HPLC) and fractions were analyzed with PTH assays having (1 to 4) and (12 to 23) epitopes, respectively. The serum of each patient was similarly analyzed. Peaks of immunoreactivity identified were submitted to sequence analysis to recover the S-35-methionine residues in positions 8 and 18. Results. Three regions of interest were identified with PTH assays. They corresponded to non-(1-84) PTH fragments (further divided in regions 3 and 4), a peak of N-PTH migrating in front of hPTH (1-84) (region 2) and a peak of immunoreactivity corresponding to the elution position of hPTH (1-84) (region 1). The last corresponded to a single sequence starting at position 1. Region 2 gave similar results in all cases (a major signal starting at position 1) but also sometimes minor sequences starting at position 4 or 7. Regions 3 and 4 always identified a major sequence starting at positions 7 and minor sequences starting at positions 8, 10, and 15. Surprisingly, a major signal starting at position 1 was also present in region 3. The HPLC profile obtained from a given patient's parathyroid cells was qualitatively similar to the one obtained with his/her serum in each case. Conclusion. These results indicate that non-(1-84) PTH fragments are composed of a family of fragments which may be generated by specific or progressive cleavage at the N region. The longest fragment starts at position 4 and the shortest at position 15. A peptide starting at position 7 appears as the major component of non-(1-84) PTH fragments. The generation process is similar to the one described for smaller C-PTH fragments a number of years ago, suggesting a similar production mechanism and source for all C-PTH fragments.