Circulating PTH molecular forms: What we know and what we don't
2006; Elsevier BV; Volume: 70; Linguagem: Inglês
10.1038/sj.ki.5001599
ISSN1523-1755
Autores Tópico(s)Fibroblast Growth Factor Research
ResumoCirculating parathyroid hormone (PTH) molecular forms have been identified by three generations of PTH assays after gel chromatography or high-performance liquid chromatography fractionation of serum. Carboxyl-terminal (C) fragments missing the amino-terminal (N) structure of PTH(1–84) were identified first. They represent 80% of circulating PTH in normal individuals and up to 95% in renal failure patients. They are regulated by calcium (Ca) slightly differently than PTH(1–84), occurring in a relatively smaller proportion relative to the latter in hypocalcemia but in a much larger proportion in hypercalcemia. Synthetic C-PTH fragments do not bind to the PTH/PTHrP type I receptor and are not implicated in the classical biological effect of PTH(1–84). They bind to a different C-PTH receptor and exert biological actions on bone that are opposite to those of PTH(1–84). The integrity of the distal C-structure appears to be important for these biological effects, and it is uncertain if all C-PTH fragments are intact up to position 84. A second category of C-PTH fragment has a partially preserved N-structure. They are called non-(1–84) PTH or N-truncated fragments. They react in Intact (I)-PTH assays but not in PTH assays with a 1–4 epitope. They are acutely regulated by Ca2+ concentration. They also exert similar hypocalcemic and antiresorptive effects but have 10-fold greater affinity for the C-PTH receptor compared to other C-PTH fragments. Even if they represent only 10% of all C-PTH fragments, they could be as relevant biologically. An N form of PTH other than PTH(1–84) has been identified in the circulation. It reacts very well in PTH assays with a 1–4 epitope but poorly in I-PTH assay with a 12–18 epitope. It is oversecreted in severe primary and secondary hyperparathyroidism and in parathyroid cancers. Its biological activity is still unknown. Overall, these studies suggest that PTH(1–84) and C-PTH fragments are regulated differently to exert opposite biological effects on bone via two different receptors. This may serve to control bone turnover and Ca concentration more efficiently. Circulating parathyroid hormone (PTH) molecular forms have been identified by three generations of PTH assays after gel chromatography or high-performance liquid chromatography fractionation of serum. Carboxyl-terminal (C) fragments missing the amino-terminal (N) structure of PTH(1–84) were identified first. They represent 80% of circulating PTH in normal individuals and up to 95% in renal failure patients. They are regulated by calcium (Ca) slightly differently than PTH(1–84), occurring in a relatively smaller proportion relative to the latter in hypocalcemia but in a much larger proportion in hypercalcemia. Synthetic C-PTH fragments do not bind to the PTH/PTHrP type I receptor and are not implicated in the classical biological effect of PTH(1–84). They bind to a different C-PTH receptor and exert biological actions on bone that are opposite to those of PTH(1–84). The integrity of the distal C-structure appears to be important for these biological effects, and it is uncertain if all C-PTH fragments are intact up to position 84. A second category of C-PTH fragment has a partially preserved N-structure. They are called non-(1–84) PTH or N-truncated fragments. They react in Intact (I)-PTH assays but not in PTH assays with a 1–4 epitope. They are acutely regulated by Ca2+ concentration. They also exert similar hypocalcemic and antiresorptive effects but have 10-fold greater affinity for the C-PTH receptor compared to other C-PTH fragments. Even if they represent only 10% of all C-PTH fragments, they could be as relevant biologically. An N form of PTH other than PTH(1–84) has been identified in the circulation. It reacts very well in PTH assays with a 1–4 epitope but poorly in I-PTH assay with a 12–18 epitope. It is oversecreted in severe primary and secondary hyperparathyroidism and in parathyroid cancers. Its biological activity is still unknown. Overall, these studies suggest that PTH(1–84) and C-PTH fragments are regulated differently to exert opposite biological effects on bone via two different receptors. This may serve to control bone turnover and Ca concentration more efficiently. PTH(1–84) is the main synthetic product of parathyroid cells in most species.1.Kronenberg H.M. Bringhurst R.F. Segre G.V. Potts Jr, J.T. Parathyroid hormone. Biosynthesis and metabolism.in: Bilezikian J. Marcus R. Levine M.A. The Parathyroids. Academic Press, New York2001: 17-30Crossref Google Scholar It exerts its classical biological effects on bone and kidney through its first 34 amino acids, that is, its amino-terminal (N) structure.2.Tregear G.W. Van Rietschoten J. Greene E. et al.Bovine parathyroid hormone: minimum chain length of synthetic peptide required for biological activity.Endocrinology. 1973; 93: 1349-1353Crossref PubMed Scopus (274) Google Scholar The latter binds to the type I PTH/PTHrP receptor and activates both the protein kinase A and C pathways in target tissues.3.Abou-Samra A.B. Jüppner H. Force T. et al.Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol trisphosphates and increases intracellular free calcium.Proc Natl Acad Sci USA. 1992; 89: 2732-2736Crossref PubMed Scopus (1000) Google Scholar The carboxyl-terminal (C) structure of PTH(1–84), the last 50 amino acids, has no known direct influence on this receptor.4.Rosenblatt M. Segre G.V. Tregear G.W. et al.Human parathyroid hormone: synthesis and chemical, biological, and immunological evaluation of the carboxyl-terminal region.Endocrinology. 1978; 103: 978-984Crossref PubMed Scopus (27) Google Scholar, 5.Pines M. Adams A.E. Stueckle S. et al.Generation and characterization of human kidney cell lines stably expressing recombinant human PTH/PTHrP receptor: lack of interaction with a C-terminal human PTH peptide.Endocrinology. 1994; 135: 1713-1716Crossref PubMed Scopus (67) Google Scholar This biological reality poorly reflects the composition of circulating PTH in humans and animals, as it is mainly comprised of C-PTH fragments,6.Habener J.F. Segre G.V. Powell D. et al.Immunoreactive parathyroid hormone in circulation of man.Nature (New Biol). 1972; 238: 152-154Crossref PubMed Scopus (48) Google Scholar, 7.Segre G.V. Habener J.F. Powell D. et al.Parathyroid hormone in human plasma. Immunochemical chracterization and biological implications.J Clin Invest. 1972; 51: 3163-3172Crossref PubMed Google Scholar constituting a major paradox that is just beginning to be resolved. The quantity of C-PTH fragments relative to that of PTH(1–84) is both acutely and chronically well-regulated, which has biological implications.8.D'Amour P. Effects of acute and chronic hypercalcemia on parathyroid function and circulating parathyroid hormone molecular forms.Eur J Endocrinol. 2002; 148: 407-410Crossref Scopus (20) Google Scholar These have been difficult to uncover because a less well-characterized, not yet cloned receptor appears to be involved.9.Murray T.M. Rao L.G. Divieti P. Bringhurst F.R. Parathyroid hormone secretion and action: evidence for discrete receptors for the carboxyl-terminal region and related biological actions of carboxyl-terminal ligands.Endocr Rev. 2005; 26: 78-113Crossref PubMed Scopus (240) Google Scholar The central theme of our presentation is what we know and do not know about circulating PTH molecular forms and their biological effects through two different PTH receptors. Two main findings, that PTH(1–84) biological effects were only dependent on its N-structure2.Tregear G.W. Van Rietschoten J. Greene E. et al.Bovine parathyroid hormone: minimum chain length of synthetic peptide required for biological activity.Endocrinology. 1973; 93: 1349-1353Crossref PubMed Scopus (274) Google Scholar and that circulating PTH was mainly composed of C-PTH fragments,6.Habener J.F. Segre G.V. Powell D. et al.Immunoreactive parathyroid hormone in circulation of man.Nature (New Biol). 1972; 238: 152-154Crossref PubMed Scopus (48) Google Scholar, 7.Segre G.V. Habener J.F. Powell D. et al.Parathyroid hormone in human plasma. Immunochemical chracterization and biological implications.J Clin Invest. 1972; 51: 3163-3172Crossref PubMed Google Scholar have influenced the evolution of PTH assays. They were believed to limit the capacity of PTH measurements to reflect the true biological activity of the hormone with radio-immunoassays (RIAs) having epitopes in the C-structure of the hormone, such as mid- and C-terminal PTH RIAs. This was particularly true in terminal renal failure patients, where these fragments accumulate and represent more than 95% of the hormone in the circulation,10.Brossard J.H. Cloutier M. Roy L. et al.Accumulation of non-(1–84) molecular form of parathyroid hormone (PTH) detected by intact PTH assay in renal failure: importance in the interpretation of PTH values.J Clin Endocrinol Metab. 1996; 81: 3923-3929Crossref PubMed Scopus (281) Google Scholar complicating correlation analysis with any measurements reflecting PTH(1–84) biological effects.11.Cohen-Solal M.E. Sebert J.L. Boudailliez B. et al.Comparison of intact, midregion and carboxyl-terminal assays of parathyroid hormone for the diagnosis of bone disease in hemodialyzed patients.J Clin Endocrinol Metab. 1991; 73: 510-514Google Scholar This has dictated the development of a second generation of PTH assay, Intact (I)-PTH assay, which uses solid-phase capture antibodies purified by affinity chromatography against hPTH(39–84) and revealing antibodies purified against hPTH(1–34). Most of the latter have their main epitope in region 13–34 of the PTH(1–34) structure, early epitopes like 12–18 and 13–24 being more frequent than distal epitope 26–32.12.D'Amour P. Brossard J.H. Räkel A. et al.Evidence that the amino-terminal composition of non-(1–84) parathyroid hormone fragments starts before position 19.Clin Chem. 2005; 51: 169-176Crossref PubMed Scopus (50) Google Scholar These assays do not detect mid- or C-PTH fragments missing a N-structure, and were believed to react only with hPTH(1–84). They had a major impact in the PTH field both clinically and experimentally, allowing the comparison of quantitative results between laboratories for the first time.13.Felsenfeld A.J. Llach F. Parathyroid gland function in chronic renal failure.Kidney Int. 1993; 43: 771-789Abstract Full Text PDF PubMed Scopus (148) Google Scholar, 14.Goodman W.G. Belin T.R. Saluski I.B. In vivo assessments of calcium-regulated parathyroid hormone release in secondary hyperparathyroidism.Kidney Int. 1996; 50: 1834-1844Abstract Full Text PDF PubMed Scopus (19) Google Scholar They were eventually demonstrated to react with C-PTH fragments having a partially preserved N-structure, also called non-(1–84) PTH fragments or N-truncated C-fragments, by my laboratory.10.Brossard J.H. Cloutier M. Roy L. et al.Accumulation of non-(1–84) molecular form of parathyroid hormone (PTH) detected by intact PTH assay in renal failure: importance in the interpretation of PTH values.J Clin Endocrinol Metab. 1996; 81: 3923-3929Crossref PubMed Scopus (281) Google Scholar These fragments accumulate and represent up to 45% of the immunoreactivity in renal failure patients, but only 20% in normal individuals.10.Brossard J.H. Cloutier M. Roy L. et al.Accumulation of non-(1–84) molecular form of parathyroid hormone (PTH) detected by intact PTH assay in renal failure: importance in the interpretation of PTH values.J Clin Endocrinol Metab. 1996; 81: 3923-3929Crossref PubMed Scopus (281) Google Scholar Since this was believed to impair their capacity to appreciate hPTH(1–84) biological effects in vivo, particularly in terminal renal failure patients,15.Quarles L.D. Lobaugh B. Murphy G. Intact parathyroid hormone overestimates the presence and severity of parathyroid-mediated osseous abnormalities in uremia.J Clin Endocrinol Metab. 1992; 75: 145-150Crossref PubMed Scopus (0) Google Scholar a third generation of PTH assays was developed. It uses the same capture antibodies as second-generation assays, but revealing antibodies with a 1–4 epitope.16.John M.R. Coolman W.G. Gao P. et al.A novel immunoradiometric assay detects full-length human PTH but not amino-terminally truncated fragments: implications for PTH measurements in renal failure.J Clin Endocrinol Metab. 1999; 84: 4287-4290Crossref PubMed Google Scholar, 17.Gao P. Scheibel S. D'Amour P. et al.Development of a novel immunoradiometric assay exclusively for biologically active whole parathyroid hormone 1–84: implications for improvement of accurate assessment of parathyroid function.J Bone Miner Res. 2001; 16: 605-614Crossref PubMed Scopus (335) Google Scholar This eliminates the immunoreactivity associated with hPTH(7–84) or non-(1–84) PTH fragments in the second generation of PTH assays.16.John M.R. Coolman W.G. Gao P. et al.A novel immunoradiometric assay detects full-length human PTH but not amino-terminally truncated fragments: implications for PTH measurements in renal failure.J Clin Endocrinol Metab. 1999; 84: 4287-4290Crossref PubMed Google Scholar, 17.Gao P. Scheibel S. D'Amour P. et al.Development of a novel immunoradiometric assay exclusively for biologically active whole parathyroid hormone 1–84: implications for improvement of accurate assessment of parathyroid function.J Bone Miner Res. 2001; 16: 605-614Crossref PubMed Scopus (335) Google Scholar These assays were believed to be specific for hPTH(1–84) until we again demonstrated that they also react with an N-form of PTH distinct from hPTH(1–84) on serum PTH high-performance liquid chromatography (HPLC) profiles obtained in normal individuals and in patients with primary or secondary hyperparathyroidism.18.D'Amour P. Brossard J.H. Rousseau L. et al.Amino-terminal form of parathyroid hormone (PTH) with immunologic similarities to hPTH(1–84) is overproduced in primary and secondary hyperparathyroidism.Clin Chem. 2003; 49: 2037-2044Crossref PubMed Scopus (94) Google Scholar This molecular form of PTH is poorly reactive in I-PTH assays with early epitopes covering region 15–20 but fully reactive in I-PTH assays with a distal epitope, suggesting some modification in region 15–20.12.D'Amour P. Brossard J.H. Räkel A. et al.Evidence that the amino-terminal composition of non-(1–84) parathyroid hormone fragments starts before position 19.Clin Chem. 2005; 51: 169-176Crossref PubMed Scopus (50) Google Scholar, 18.D'Amour P. Brossard J.H. Rousseau L. et al.Amino-terminal form of parathyroid hormone (PTH) with immunologic similarities to hPTH(1–84) is overproduced in primary and secondary hyperparathyroidism.Clin Chem. 2003; 49: 2037-2044Crossref PubMed Scopus (94) Google Scholar A representative illustration of PTH molecular forms detected by three generations of PTH assays is presented in Figure 1. C-PTH fragments, with or without a partially preserved N-structure, are secreted by parathyroid glands in a calcium-dependent manner10.Brossard J.H. Cloutier M. Roy L. et al.Accumulation of non-(1–84) molecular form of parathyroid hormone (PTH) detected by intact PTH assay in renal failure: importance in the interpretation of PTH values.J Clin Endocrinol Metab. 1996; 81: 3923-3929Crossref PubMed Scopus (281) Google Scholar, 19.Mayer G.P. Keaton J.A. Hurst J.G. Habener J.F. 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Lecavalier L. et al.Influence of serum Ca concentration on circulating molecular forms of PTH in three species.Am J Physiol. 1986; 251: E680-E687PubMed Google Scholar, 29.D'Amour P. Palardy J. Bashali G. et al.Modulation of circulating parathyroid hormone immunoheterogeneity in man by ionized calcium concentration.J Clin Endocrinol Metab. 1992; 75: 525-532Google Scholar This regulation operates within the normal range of Ca2+ values in humans.30.D'Amour P. Räkel A. Brossard J.H. et al.Acute regulation of circulating PTH molecular forms by calcium. Utility of PTH fragments/PTH(1–84) ratios derived from 3 generations of PTH assays.J Clin Endocrinol Metab. 2006; 91: 283-289Crossref PubMed Scopus (43) Google Scholar While stimulating PTH secretion, hypocalcemia favors the output of PTH(1–84) over that of C-PTH fragments, decreasing the C-PTH fragments/PTH(1–84) ratio to its lowest value.10.Brossard J.H. Cloutier M. Roy L. et al.Accumulation of non-(1–84) molecular form of parathyroid hormone (PTH) detected by intact PTH assay in renal failure: importance in the interpretation of PTH values.J Clin Endocrinol Metab. 1996; 81: 3923-3929Crossref PubMed Scopus (281) Google Scholar, 28.D'Amour P. Labelle F. Lecavalier L. et al.Influence of serum Ca concentration on circulating molecular forms of PTH in three species.Am J Physiol. 1986; 251: E680-E687PubMed Google Scholar, 29.D'Amour P. Palardy J. Bashali G. et al.Modulation of circulating parathyroid hormone immunoheterogeneity in man by ionized calcium concentration.J Clin Endocrinol Metab. 1992; 75: 525-532Google Scholar, 30.D'Amour P. Räkel A. Brossard J.H. et al.Acute regulation of circulating PTH molecular forms by calcium. Utility of PTH fragments/PTH(1–84) ratios derived from 3 generations of PTH assays.J Clin Endocrinol Metab. 2006; 91: 283-289Crossref PubMed Scopus (43) Google Scholar While inhibiting PTH secretion, hypercalcemia favors the output of C-PTH fragments over that of PTH(1–84), increasing the C-PTH fragments/PTH(1–84) ratio to its highest value.10.Brossard J.H. Cloutier M. Roy L. et al.Accumulation of non-(1–84) molecular form of parathyroid hormone (PTH) detected by intact PTH assay in renal failure: importance in the interpretation of PTH values.J Clin Endocrinol Metab. 1996; 81: 3923-3929Crossref PubMed Scopus (281) Google Scholar, 28.D'Amour P. Labelle F. Lecavalier L. et al.Influence of serum Ca concentration on circulating molecular forms of PTH in three species.Am J Physiol. 1986; 251: E680-E687PubMed Google Scholar, 29.D'Amour P. Palardy J. Bashali G. et al.Modulation of circulating parathyroid hormone immunoheterogeneity in man by ionized calcium concentration.J Clin Endocrinol Metab. 1992; 75: 525-532Google Scholar, 30.D'Amour P. Räkel A. Brossard J.H. et al.Acute regulation of circulating PTH molecular forms by calcium. Utility of PTH fragments/PTH(1–84) ratios derived from 3 generations of PTH assays.J Clin Endocrinol Metab. 2006; 91: 283-289Crossref PubMed Scopus (43) Google Scholar This applies to both types of C-PTH fragments.10.Brossard J.H. Cloutier M. Roy L. et al.Accumulation of non-(1–84) molecular form of parathyroid hormone (PTH) detected by intact PTH assay in renal failure: importance in the interpretation of PTH values.J Clin Endocrinol Metab. 1996; 81: 3923-3929Crossref PubMed Scopus (281) Google Scholar, 30.D'Amour P. Räkel A. Brossard J.H. et al.Acute regulation of circulating PTH molecular forms by calcium. Utility of PTH fragments/PTH(1–84) ratios derived from 3 generations of PTH assays.J Clin Endocrinol Metab. 2006; 91: 283-289Crossref PubMed Scopus (43) Google Scholar Apart from the acute regulation of circulating PTH molecular forms, further chronic regulation can be demonstrated based on PTH(1–84) needs. When more PTH is needed to maintain Ca2+ concentration, as in evolving secondary hyperparathyroidism due to vitamin D deficiency,31.Cloutier M. Gascon-Barré M. D'Amour P. Chronic adaptation of dog parathyroid function to a low calcium high sodium, vitamin D deficient diet.J Bone Miner Res. 1992; 7: 1021-1028Crossref PubMed Scopus (46) Google Scholar renal failure10.Brossard J.H. Cloutier M. Roy L. et al.Accumulation of non-(1–84) molecular form of parathyroid hormone (PTH) detected by intact PTH assay in renal failure: importance in the interpretation of PTH values.J Clin Endocrinol Metab. 1996; 81: 3923-3929Crossref PubMed Scopus (281) Google Scholar or half parathyroidectomy,32.Cloutier M. Gagnon Y. Brossard J.H. et al.Adaptation of parathyroid function to IV 1,25-dihydroxyvitamin D3 or partial parathyroidectomy in normal dogs.J Endocrinol. 1997; 155: 133-141Crossref PubMed Scopus (20) Google Scholar more PTH(1–84) than C-PTH fragments is secreted at all Ca2+ concentrations, resulting in a lower C-PTH fragments/PTH(1–84) ratio than initially present.10.Brossard J.H. Cloutier M. Roy L. et al.Accumulation of non-(1–84) molecular form of parathyroid hormone (PTH) detected by intact PTH assay in renal failure: importance in the interpretation of PTH values.J Clin Endocrinol Metab. 1996; 81: 3923-3929Crossref PubMed Scopus (281) Google Scholar, 31.Cloutier M. Gascon-Barré M. D'Amour P. Chronic adaptation of dog parathyroid function to a low calcium high sodium, vitamin D deficient diet.J Bone Miner Res. 1992; 7: 1021-1028Crossref PubMed Scopus (46) Google Scholar, 32.Cloutier M. Gagnon Y. Brossard J.H. et al.Adaptation of parathyroid function to IV 1,25-dihydroxyvitamin D3 or partial parathyroidectomy in normal dogs.J Endocrinol. 1997; 155: 133-141Crossref PubMed Scopus (20) Google Scholar The same is true when less PTH(1–84) is needed, as in chronic nonparathyroid hypercalcemia,33.Brossard J.H. Whittom S. Lepage R. D'Amour P. Carboxyl-terminal fragments of parathyroid hormone are not secreted preferentially in primary hyperparathyroidism as they are in other causes of hypercalcemia.J Clin Endocrinol Metab. 1993; 77: 413-419Crossref PubMed Scopus (0) Google Scholar during vitamin D replacement therapy in vitamin D-deficient animals34.Cloutier M. Brossard J.H. Gascon-Barré M. D'Amour P. Lack of involution of hyperplastic parathyroid glands in dogs. Adaptation via a decrease in the calcium stimulation set point and a change in secretion profile.J Bone Miner Res. 1994; 9: 621-629Crossref PubMed Scopus (35) Google Scholar and during 1,25(OH)2D administration in dogs32.Cloutier M. Gagnon Y. Brossard J.H. et al.Adaptation of parathyroid function to IV 1,25-dihydroxyvitamin D3 or partial parathyroidectomy in normal dogs.J Endocrinol. 1997; 155: 133-141Crossref PubMed Scopus (20) Google Scholar; less PTH(1–84) than C-PTH fragments is secreted at all Ca2+ concentrations, culminating in a higher C-PTH fragments/PTH(1–84) ratio than initially present.32.Cloutier M. Gagnon Y. Brossard J.H. et al.Adaptation of parathyroid function to IV 1,25-dihydroxyvitamin D3 or partial parathyroidectomy in normal dogs.J Endocrinol. 1997; 155: 133-141Crossref PubMed Scopus (20) Google Scholar, 33.Brossard J.H. Whittom S. Lepage R. D'Amour P. Carboxyl-terminal fragments of parathyroid hormone are not secreted preferentially in primary hyperparathyroidism as they are in other causes of hypercalcemia.J Clin Endocrinol Metab. 1993; 77: 413-419Crossref PubMed Scopus (0) Google Scholar, 34.Cloutier M. Brossard J.H. Gascon-Barré M. D'Amour P. Lack of involution of hyperplastic parathyroid glands in dogs. Adaptation via a decrease in the calcium stimulation set point and a change in secretion profile.J Bone Miner Res. 1994; 9: 621-629Crossref PubMed Scopus (35) Google Scholar This tight regulation of circulating C-PTH fragments has some kind of biological implication. C-PTH fragments produced during the peripheral metabolism of 125I-bPTH(1–84) in dogs35.Segre G.V. Niall H.D. Sauer R.T. Potts Jr, J.T. Edman degradation of radioiodinated parathyroid hormone: application to sequence analysis and hormone metabolism in vivo.Biochemistry. 1977; 16: 2417-2427Crossref PubMed Scopus (36) Google Scholar and rats36.Segre G.V. D'Amour P. Potts Jr, J.T. Metabolism of radioiodinated bovine parathyroid hormone in the rat.Endocrinology. 1976; 99: 1645-1652Crossref PubMed Scopus (61) Google Scholar start their N-structure at positions 34, 37, 41 and 43 of the bPTH structure. Porcine parathyroid cells also secrete internally labelled C-PTH fragments starting at positions 34 and 37.37.Morrissey J.J. Hamilton J.W. MacGregor R.R. Cohn D.V. The secretion of parathormone fragments 34–84 and 37–84 by dispersed porcine parathyroid cells.Endocrinology. 1980; 107: 164-171Crossref PubMed Scopus (67) Google Scholar The integrity of the C-terminal end of these fragments remains an open issue. C-PTH RIAs detect hPTH(53–83) as well as hPTH(53–84), and we do not know how many amino acids must be removed from the C-terminal end to impair immunoreactivity in these assays. This is a practical issue, because removing glutamine in position 84 can impair biological activity38.Takasu H. Baba H. Inomata N. et al.The 69–84 amino acid region of the parathyroid hormone molecule is essential for the interaction of the hormone with the binding sites with carboxyl-terminal specificity.Endocrinology. 1996; 137: 5537-5543Crossref PubMed Scopus (36) Google Scholar or greatly reduce affinity for the C-PTH receptor.39.Divieti P. Geller A.I. Suliman G. et al.Receptors specific for the carboxyl-terminal region of parathyroid hormone on bone-derived cells: determinants of ligand binding and bioactivity.Endocrinology. 2005; 146: 1863-1870Crossref PubMed Scopus (66) Google Scholar We have studied the N-structure of non-(1–84) PTH fragments labelled internally with 35S-methionine and secreted by parathyroid cells from patients with primary or secondary hyperparathyroidism.40.D'Amour P. Brossard J.H. Rousseau L. et al.Structure of non-(1–84) PTH fragments secreted by parathyroid glands in primary and secondary hyperparathyroidism.Kidney Int. 2005; 68: 998-1007Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar The N-structure of these fragments started at positions 4, 7, 8, 10 and 15 of the hPTH(1–84) structure, positions 7 and 15 being dominant. We have also studied the C-terminal end of these fragments, using an antibody provided by Immutopics International, which detected hPTH(53–84) better than hPTH(53–83). Comparing the recovery of non(1–84) PTH fragments obtained with this assay to that of an I-PTH assay in HPLC profiles, we could conclude that not all non-(1–84) PTH fragments had an intact C-terminal end structure.40.D'Amour P. Brossard J.H. Rousseau L. et al.Structure of non-(1–84) PTH fragments secreted by parathyroid glands in primary and secondary hyperparathyroidism.Kidney Int. 2005; 68: 998-1007Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar More studies with better C-terminal end antibodies are required to confirm these findings. C-PTH fragments, with or without a partially preserved N-structure, do not interact with the type I PTH/PTHrP receptor4.Rosenblatt M. Segre G.V. Tregear G.W. et al.Human parathyroid hormone: synthesis and chemical, biological, and immunological evaluation of the carboxyl-terminal region.Endocrinology. 1978; 103: 978-984Crossref PubMed Scopus (27) Google Scholar, 5.Pines M. Adams A.E. Stueckle S. et al.Generation and characterization of human kidney cell lines stably expressing recombinant human PTH/PTHrP receptor: lack of interaction with a C-terminal human PTH peptide.Endocrinology. 1994; 135: 1713-1716Crossref PubMed Scopus (67) Google Scholar, 41.Nguyen-Yamamoto L. Rousseau L. Brossard J.H. et al.Synthetic carboxyl-terminal fragments of PTH decrease ionized calcium concentration in rats by acting on a receptor different from the PTH/PTHrP receptor.Endocrinology. 2001; 142: 136-139Crossre
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