Detection and characterization of small midregion parathyroid hormone fragment(s) in normal and hyperparathyroid glands and sera by immunoextraction and region-specific radioimmunoassays

We have employed region-specific RIAs directed against amino-terminal (N), midregion (M), or carboxyl-terminal (C) regions of parathyroid hormone (PTH) to analyze glandular and circulating PTH species in normal subjects and in persons with primary hyperparathyroidism. Immunoaffinity methods have been developed for validation of RIA measurements and for extraction of circulating PTH spcies. Chromatographic analyses of normal and adenomatous parathyroid-gland extracts and electrophoretic analyses of parathyroid-gland immunoextracts revealed at least four PTH species, each with a distinct pattern of cross-reactivity in the sequence-specific RIAs. Intact PTH (mol wt, 9500) was detected by each type of RIA. The large carboxyl-fragment (mol wt, 5500), was detected only by M and C RIAs. A somewhat smaller fragment (mol wt, 4000) was identified that was detected by M RIA but not by C RIA. We also detected in parathyroid extracts a small N-fragment (mol wt, 3400), which had no reactivity in M or C RIAs. In normal and hyperparathyroid plasmas the predominant PTH species is a large C-fragment (mol wt, 5500) with cross-reactivity in both M and C RIAs. N RIA results indicated relatively low levels of intact PTH or small N-fragments in immunoextracts of peripheral plasmas. However, plasmas from persons with primary hyperparathyroidism did contain an additional prominent PTH species (mol wt, 4000) that was similar to M-fragment(s) found in parathyroid-gland extracts. Since small M-fragment(s) cross-reacts only with the M-antisera, we were able to develop, for isolation and measurement of the fragment(s), a sequential immunoextraction procedure that does not use chromatography. Using this procedure we found that normal basal sera have no detectable small M-fragment(s). However, circulating M-fragment(s) is detectable in normal persons during EDTA-induced hypocalcemial; the relative increase in M-fragment(s) is three times greater than that observed for total anti-C immunoreactivity, which includes large C-fragment(s) and intact PTH. The accumulation of circulating small M-fragment(s) in hypercalcemic hyperparathyroid persons and in normal persons during EDTA-induced hypocalcemia suggest that it may somehow indicate the level of parathyroid secretory activity. Since a small M-species in parathyroid tissues has size and immunochemical characteristics similar to circulating M-fragment(s), this species might be secreted by parathyroid tissue.