Circulating forms of parathyroid hormone: peeling back the onion.

Circulating parathyroid hormone (PTH) regulates normal bone and mineral ion homeostasis and is central in the pathogenesis of bone disease in primary and secondary hyperparathyroidism, especially in advanced renal insufficiency (1 ). The major biological effects of PTH, a linear polypeptide 84 amino acids in length, reflect activation of the G-protein-coupled PTH/PTHrP receptor (PTHR) expressed on target cells in the renal tubules and the osteoblastic lineage of bone (2 ). Activation of PTHRs requires only the most N-terminal portion of PTH [PTH(1–34) is a full agonist] and is mostly eliminated if the N-terminal serine residue of the human hormone is removed. PTH, secreted by the parathyroid glands primarily in response to changes in blood ionized calcium, undergoes rapid endopeptidic cleavage, mainly by Kupffer cells in the liver (3 ). This event generates a series of N-truncated “CPTH” fragments (the corresponding N-terminal segments are destroyed in situ) that reenter the circulation and are cleared mainly by glomerular filtration, an important route for PTH clearance as well (4 ). Chemical analysis of PTH fragments isolated from rat blood after administration of radiolabeled bovine PTH established that hepatic metabolism yields a family of CPTH fragments with NH2 termini ranging between residues 34 and 43, although the methods used likely would not have detected substantially larger CPTH fragments (5 ). Interestingly, chemically similar CPTH fragments are secreted directly from the parathyroid glands as well, and the ratio of secreted CPTH fragments to intact PTH increases as blood calcium increases (6, 7). CPTH fragments typically circulate at a threeto fivefold molar excess over intact PTH, but this ratio may increase dramatically in end-stage renal disease (ESRD), depending on the severity and chronicity of renal insufficiency and the blood concentrations of calcium, phosphate, and 1,25-dihydroxyvitamin D, all of which regulate PTH secretion (1, 8). Measurement of circulating biologically active PTH is crucial for the differential diagnosis of hypercalcemia and hypocalcemia and in the diagnosis and management of primary hyperparathyroidism, vitamin D deficiency, and renal osteodystrophy. Early efforts to measure PTH by use of single-antibody RIAs directed against the more immunogenic C-terminal region of PTH were confounded by the high and variable amounts of reactive CPTH fragments, especially in ESRD, in which results of PTH assays correlated poorly with bone histology (9 ). In fact, observations that RIAs based on different antisera produced divergent results had provided early clues to the surprising heterogeneity of circulating PTH peptides (10 ). This problem appeared to be solved with the advent of “intact PTH” two-site IRMAs, which typically use a solid-phase capture antibody directed against the PTH COOH terminus [usually PTH(39–84)] and a labeled detection antibody raised against PTH(1–34) (11 ) (see Fig. 1). These IRMAs thus were “blind” to the major “short” CPTH fragments previously identified in blood and reacted exclusively with peptides long enough to incorporate both Cand N-terminal PTH sequences, the only known candidate for which was intact PTH(1–84). The clinical utility of these first-generation IRMAs in the diagnosis of primary hyperparathyroidism and in monitoring the severity of secondary hyperparathyroidism in ESRD is well established. On the other hand, it is known that serum concentrations of intact PTH measured in this way tend to overestimate the severity of PTH-related bone disease seen on biopsy in ESRD and that a decrease in intact PTH concentrations to normal in this setting, as with calcium and active vitamin D analog therapy, is associated with adynamic bone disease (1 ). This discrepancy was attributed to the resistance of bone to PTH in ESRD, and nephrologists circumvented the problem simply by targeting intact PTH to concentrations twoto fourfold above normal (1, 12). All seemed well until 1996, when Brossard et al. (13 ) reported that up to one-half of the signal detected by intact PTH assays in blood from dialyzed ESRD patients is attributable to circulating forms of PTH that can be separated from hPTH(1–84) by reversed-phase HPLC. These peptides, collectively termed “non-(1–84) PTH”, subsequently were found not to react in new, secondgeneration PTH IRMAs, in which the recognition epitope of the detection antibody is located at the extreme NH2 terminus of PTH (14 ). Thus, peptides lacking the Nterminal serine of hPTH fail to register in these new assays, whereas the detection antibodies used in the first-generation IRMAs bind N-truncated peptides, such as PTH(7–84), just as well as those with an intact NH2 terminus (14, 15). The precise chemical structures of these N-truncated non-(1–84) PTH fragments remain unknown, although they are long enough to react with both Cand N-directed antibodies and eluted similarly to synthetic hPTH(7–84) in the original HPLC systems used (16 ).