THE DEVELOPMENT OF improved assays for the measurement

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1 X/03/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 88(10): Printed in U.S.A. Copyright 2003 by The Endocrine Society doi: /jc Clinical Utility of an Immunoradiometric Assay for Parathyroid Hormone (1 84) in Primary Hyperparathyroidism SHONNI J. SILVERBERG, PING GAO, IJEOMA BROWN, PAUL LOGERFO, TOM L. CANTOR, AND JOHN P. BILEZIKIAN Departments of Medicine (S.J.S., I.B., J.P.B.), Pharmacology (J.P.B.), and Surgery (P.L.), College of Physicians and Surgeons, Columbia University, New York, New York 10032; and Scantibodies Laboratory (P.G., T.L.C.), Santee, California The reliable diagnosis of primary hyperparathyroidism depends on the measurement of PTH. The PTH assays in widespread use measure not only the hormone but also hormone fragments, thus limiting the clinical utility of the assays. A new immunoradiometric assay (IRMA) using an antigenic determinant at the extreme amino-terminal of the PTH molecule detects only full-length PTH (1 84). We compared three PTH assays and determined the presence of PTH (1 84) and PTH fragments in serum and parathyroid adenomas of patients with primary hyperparathyroidism. We studied 56 patients with primary hyperparathyroidism. PTH levels were increased in 63% using the midmolecule RIA; in 73% in the intact IRMA; and in 96% in the PTH (1 84)-IRMA. The PTH (1 84)-IRMA correlated with the other assays (midmolecule RIA R 0.736; P < ; intact -IRMA R 0.951; P < ) and indices of disease activity (serum calcium R THE DEVELOPMENT OF improved assays for the measurement of PTH in the circulation has had a significant impact on the diagnosis and understanding of parathyroid gland dysfunction (1 5). However, efforts in this regard have been hampered by the presence of PTH fragments (5 8). PTH assays commonly measured these fragments, thus confounding attempts to determine true levels of bioactive hormone. Inactive carboxy-terminal fragments of PTH are generated by metabolism of hormone in the circulation, within the liver, in the parathyroid gland itself, and conceivably in other organs (5, 6, 9 12). These fragments are eliminated primarily by the kidney. Early measurements of PTH by RIA often used antisera directed against midregion or carboxy-terminal epitopes of undefined biological activity. These assays measured active PTH as well as the carboxy-terminal fragments, posing a particular problem in patients with impaired renal function, in whom fragments typically accumulate. The introduction of the immunoradiometric assay (IRMA) offered important advantages over the RIA. Assays based on antibodies directed against epitopes on both the carboxyand the amino-terminal aspects of the PTH molecule were designed to exclude carboxy-terminal fragments from the measurements of biologically active hormone. To a certain extent, the first-generation IRMA method achieved this goal. Abbreviation: IRMA, Immunoradiometric assay , P < ; alkaline phosphatase R 0.489, P 0.001; and radius bone density R 0.366, P < 0.01). In 21 consecutive patients undergoing parathyroidectomy, 18 had parathyroid adenomas. Intact PTH was higher than PTH (1 84)- IRMA in both serum and glandular homogenates from these patients. Similar proportions of PTH (1 84) and hormone fragments were found in both adenomas [66 3% of intact PTHreflected PTH (1 84) and sera (73 2% of intact PTH reflected PTH (1 84)]. We conclude that the PTH (1 84)-IRMA offers improved diagnostic sensitivity in patients with primary hyperparathyroidism than other currently available assays. This study also provides evidence that both PTH (1 84) and PTH fragments are produced in parathyroid adenomas and that peripheral metabolism of hormone and fragment does not alter the proportion of bioactive hormone. (J Clin Endocrinol Metab 88: , 2003) However, in 1998, LePage et al. (7) demonstrated a large non-(1 84) PTH fragment that was not excluded by the intact IRMA for PTH. This large fragment comigrated with PTH (7 84) and had substantial cross-reactivity in commercially available IRMAs. It constituted as much as 50% (20 90%) of immunoreactivity by IRMA for PTH in individuals with chronic renal failure (13). A new IRMA uses affinity-purified polyclonal antibodies to the (39 84) and (1 4) amino acid regions of PTH (8, 13). Recognition of PTH in this assay requires that the entire PTH molecule must be intact, including the extreme end of the amino-terminal aspects of the PTH molecule. This assay, therefore, does not detect the large the fragment that circulates in normal patients. An assay specific for PTH (1 84) may have clinical utility in uremic patients. Renal failure patients clearly have secondary hyperparathyroidism, yet the intact -IRMA has been shown to considerably overestimate elevations in biologically active hormone concentration (7, 14, 15). In primary hyperparathyroidism, a large non-(1 84) PTH fragment is detected as well (13). In this study, the utility of this new IRMA for PTH (1 84) was assessed in a group of patients with primary hyperparathyroidism. Using the data obtained from simultaneous measurement of PTH in several assays, we investigated the presence of PTH (1 84) and PTH fragment in the adenomatous glands and in the circulation of patients with primary hyperparathyroidism. 4725

2 4726 J Clin Endocrinol Metab, October 2003, 88(10): Silverberg et al. PTH(1 84) in Primary Hyperparathyroidism PTH assays Patients and Methods The methods for the three PTH assays have been described in detail previously (1, 5, 8, 13, 16). The PTH (1 84)-IRMA [Scantibodies Laboratories, Santee, CA; henceforth referred to as PTH (1 84)-IRMA] uses two antibodies that form a detectable sandwich only if the first amino acid of PTH (1 34) and the mid- to carboxy-terminal amino acids, 39 84, of the PTH molecule are present (8). Because both amino-terminal and carboxy-terminal residues are required for detection in this assay, circulating fragments of PTH do not interfere in the assay unless present in considerable excess. The normal range for the assay using serum samples is 5 31 pg/ml, and the limit of detection is approximately 1 2 pg/ml. This normal range, determined by measuring 70 healthy subjects, is somewhat lower that the normal range established in this assay using EDTA-plasma samples, in which PTH has greater stability (17). The midmolecule RIA (henceforth referred to as midmolecule RIA) uses goat antiserum to PTH (1 84) and human [Tyr43]PTH (43 68) as tracer and standard. Intact PTH assay kits (henceforth referred to as intact IRMA) were purchased from Nichols Institute (San Juan Capistrano, CA). PTH assay comparison study Patients were recruited from the clinical and research facilities of the Metabolic Bone Diseases Unit at Columbia Presbyterian Medical Center. PTH levels were measured in the three assays described above. Serum total calcium, phosphorus, and alkaline phosphatase activity were measured by automated techniques (Technicon Instruments, Tarrytown, NY). Serum levels of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin, serum osteocalcin and urinary calcium and N-telopeptide were measured as previously described (18). Bone mineral density of the lumbar spine (L2-L4), femoral neck, and distal third of the nondominant radius was performed by dual-energy x-ray absorptiometry (Hologic Inc., Waltham, MA). Parathyroid gland study Patients with primary hyperparathyroidism referred to a single surgeon (P.L.) for parathyroidectomy were enrolled. Serum calcium levels (normal range mg/dl) were measured in a different laboratory than was used in the PTH assay comparison study (normal range mg/dl). Serum obtained from all patients immediately before surgery was frozen and batched for assay using both the PTH (1 84)-IRMA and an intact IRMA (Scantibodies Laboratories). Aliquots of parathyroid tissue were retained after a sample was sent for pathological diagnosis. Tissue specimens were processed within 30 min of parathyroidectomy, and all samples were stored at 70 C until measurements were performed. Parathyroid tissue was homogenized in 4 cc of 0.01 m cold (2 8 C) PBS in an ice bath and was centrifuged for 15 min at 1500 rpm. One percent protease inhibitor cocktail (product no. P-8340, Sigma, St. Louis, MO) was added to the homogenized tissue supernatant, which was kept at 70 C until assayed. Assays were performed on the supernatants at the same time PTH measurements were made from peripheral serum. PTH was measured in both PTH (1 84)-IRMA and intact IRMA. Serum data are presented for calcium, PTH (1 84)-IRMA and intact IRMA, and glands were assayed for PTH only, in both IRMAs. PTH as measured in the intact IRMA reflects biologically active PTH (1 84) plus large hormone fragment(s), at least one of which is likely to be PTH (7 84). The percentage of intact PTH that represents PTH (1 84) was calculated from the mean of the 21 individual patients PTH (1 84) to intact PTH ratios. Statistical analysis Data are expressed as mean sem. Correlation coefficients were used to assess concordance among the different assays for PTH as well as results obtained in analysis of glandular and serum measurements in the parathyroid gland study. Simple regression analysis assessed the association of various indices of disease activity in primary hyperparathyroidism with PTH levels as measured in the different assays. Unpaired t tests were used to compare biochemical and bone densitometric indices in those with normal and elevated PTH concentrations by the various assays, and 2 test was used to detect differences in normal and elevated values among the three assays for PTH. Paired t tests were used to compare the percentage of biologically active PTH in each patient s glandular tissue and serum. The study was approved by the Institutional Review Board of the Columbia Presbyterian Medical Center, and patients gave informed consent. Results PTH assay comparison study Fifty-six patients were included in this study (mean age 60 2 yr; 49 women and seven men). All subjects had mild primary hyperparathyroidism, with biochemical indices typical for the disease (Table 1). We compared the results of three assays for PTH in simultaneously drawn samples. Mean levels for the group in these assays were: midmolecule RIA: pg/ml (normal range 200); intact IRMA: pg/ml (normal range 10 65); PTH (1 84)-IRMA: 75 6 pg/ml (normal range 5 31). All patients had elevated serum calcium concentrations. PTH levels were above the upper limits of normal for the assay in 35 patients (63%) using the midmolecule RIA; in 41 of 56 patients (73%) measured in the intact IRMA; and in 54 (96%) patients in the PTH (1 84)-IRMA ( 2 P ; Fig. 1). The two patients with normal levels by PTH (1 84)-IRMA did not have increased PTH by any assay. One of these patients was the youngest participant in this study [20 yr of age; serum calcium 11.2 mg/dl with intact IRMA 35 pg/ml and PTH (1 84)-IRMA 27 pg/ml], in whom an adenoma was subsequently found at surgery. The other patient had levels TABLE 1. Biochemical characteristics of 56 patients with primary hyperparathyroidism Index Mean SEM Normal range Serum calcium mg/dl Serum phosphorus mg/dl Urinary calcium mg/24 h 1,25-Dihydroxy vitamin D pg/ml 25-Hydroxy vitamin D ng/ml Blood urea nitrogen mg/dl Serum creatinine mg/dl Alkaline phosphatase IU/liter Osteocalcin nmol/mmol Urinary N-telopeptide nmol/mmol creatinine FIG. 1. Simultaneous measurement of PTH levels using three different assays in 56 patients with primary hyperparathyroidism. Percentage of patients with increased levels in each assay is shown. Asterisk denotes difference in percentage of increased levels among the three assays by 2 test at P

3 Silverberg et al. PTH(1 84) in Primary Hyperparathyroidism J Clin Endocrinol Metab, October 2003, 88(10): by PTH (1 84)-IRMA at the extreme upper end of the normal range (29 pg/ml), despite persistent hypercalcemia. Elevated intact PTH was noted in this patient 1 yr after these measurements were made. In neither of these cases was the diagnosis of primary hyperparathyroidism in doubt because PTH levels were normal despite hypercalcemia. Only one patient had a suppressed PTH measurement, which was obtained in the midmolecule RIA. PTH levels in that patient were frankly elevated by intact IRMA and PTH (1 84)- IRMA methodologies. In all other cases, the normal PTH levels were in the mid- to upper reference range for the different assays. In all 14 patients who underwent parathyroidectomy, a single adenoma was removed. Patients were referred for surgery according to 1990 National Institutes of Health guidelines for parathyroidectomy in primary hyperparathyroidism (19). As expected from the guidelines (19), which are related to disease severity, those who underwent surgery had higher serum calcium levels than those who did not (surgical group mg/dl vs ; P 0.05). PTH levels were within normal limits in surgically confirmed disease in one patient in all three assays, in four patients by midmolecule RIA and intact IRMA; and in another patient by midmolecule RIA. The new PTH (1 84)-IRMA showed significant concordance with measurements made by earlier assays [midmolecule RIA R 0.736; P ; intact -IRMA R 0.951; P (Fig. 2)]. PTH (1 84)-IRMA levels also correlated with indices of disease activity in primary hyperparathyroidism. A positive correlation was found between FIG. 2. Correlation of PTH levels by PTH (1 84)-IRMA with serum calcium concentration (top panel) and with simultaneous measurements in the midmolecule RIA and intact IRMA assay for PTH (bottom panel). PTH (1 84)-IRMA and serum calcium levels [R 0.511; P (Fig. 2)] and alkaline phosphatase concentration (R 0.489; P 0.001); but values correlated negatively with bone mineral density at the site containing the greatest amount of cortical bone, the distal one third radius (bone density gram per square centimeter R 0.366; P 0.01, Z-score R 0.496; P ; and T-score R 0.450; P 0.001). The expected positive relationship was documented between PTH (1 84) IRMA and 1,25-dihydroxyvitamin D levels (R 0.340; P 0.05), but an inverse association was seen with 25-hydroxyvitamin D levels (R 0.442; P 0.005). These relationships remained significant upon reanalysis excluding the single outlier, a patient with a PTH concentration above 300 pg/ml. No relationship was found between PTH (1 84)-IRMA concentration and age, serum phosphorus, or urinary calcium excretion. Importantly, no patient had impaired renal function, and there were no associations between PTH levels in this assay and levels of blood urea nitrogen (R 0.139; P 0.368) or serum creatinine (R 0.126; P 0.414). Parathyroid gland study Twenty-one consecutive patients undergoing parathyroidectomy (aged yr; 76% female) were enrolled. Pathology revealed adenomas in 18 patients and hyperplasia in three patients. Although there were no substantial differences according to pathological diagnosis, it seems possible that the disease process, and potentially PTH metabolism, may differ in adenomatous and hyperplastic disease. Therefore, data from the three patients with parathyroid hyperplasia were excluded from this analysis. Baseline serum calcium was mg/dl (normal range ). In the adenomatous parathyroid glands, PTH levels were 1.8-fold higher in the intact IRMA than in the PTH (1 84)-IRMA (697, ,061 vs. 390, ,385 pg/ml, P 0.06), although these values were highly correlated with each other (R 0.963, P ). In serum, PTH levels were also higher (1.3-fold) by the intact IRMA, compared with measurements by the PTH (1 84)- IRMA ( pg/ml; PTH (1 84)-IRMA: pg/ml; P ), although levels of PTH in the two assays were highly correlated (R 0.961; P ). Glandular PTH levels correlated with serum PTH in the PTH (1 84)-IRMA (R 0.578, P 0.05) but not the intact IRMA. Serum calcium levels correlated with glandular PTH in both assays [PTH (1 84)-IRMA: R 0.569, P 0.05; and intact IRMA: R 0.544, P 0.05] but not with either PTH measurement in the serum. Although their data were not included in the analysis, there were no differences in serum levels of calcium or PTH or glandular PTH levels among patients with adenomas and those with hyperplastic parathyroid glands. In both parathyroid adenomas and serum in these 21 patients, most of the PTH measured in the intact IRMA reflected PTH (1 84). In the adenomas, the percentage of PTH (1 84) was calculated to be 66 3%, and the balance reflected large, previously described carboxy-terminal fragments of PTH, such as PTH (7 84) (7, 8, 13). In serum measurements, the percentage of PTH (1 84) was calculated to be 73 3%,

4 4728 J Clin Endocrinol Metab, October 2003, 88(10): Silverberg et al. PTH(1 84) in Primary Hyperparathyroidism with the rest again reflecting large carboxy-terminal fragments of PTH. The percentage of PTH (1 84) in the adenomas was not different from bioactive hormone levels in the serum of healthy subjects (paired t test P NS). Discussion In a group of patients with mild primary hyperparathyroidism, the PTH (1 84) IRMA correctly identified elevated PTH levels in 96% of clinically suspected or surgically proven cases. This represents a marked improvement on earlier assays, which revealed normal PTH concentrations in 37% by midmolecule RIA, and 27% by intact IRMA, of patients with disease. In addition to detecting PTH elevations in a significant number of patients with normal measurements by previously available assays, PTH levels detected by the PTH (1 84)-IRMA correlate strongly with known markers of disease activity. Furthermore, PTH levels in this assay are associated with the stimulatory action of PTH on renal 1- -hydroxylase (increasing 1,25-dihydroxyvitamin D3) as well as the known action of PTH excess to be catabolic at a cortical bone site, such as the distal one third radius (18, 20). This adds to clinical confidence and utility of the assay. Historically, much of the interest in the effect of circulating hormone fragments on PTH assay accuracy has come from those investigating patients with renal disease and attendant secondary hyperparathyroidism (7, 8, 20 22). In such patients, biologically inactive fragments generated by hepatic cleavage of PTH cannot undergo normal clearance by glomerular filtration. With advanced renal insufficiency, in which elevated levels of active PTH also occur, these older assays could not distinguish between the extent of secondary hyperparathyroidism and the accumulation of fragments. Although the two-site intact IRMA, using antibodies generated against epitopes near both the amino- and carboxyterminal ends of the PTH molecule, was touted as a resolution to this problem, recent data have shown this not to be the case. Samples from 112 uremic patients, using the commercially available IRMAs used in this study, revealed crossreactivity of PTH (1 84) and large non-(1 84) PTH fragments (7, 15). In patients with renal failure, measurement of this large fragment can lead to overestimation of actual PTH concentration as much as 2-fold (7, 8, 15, 23). Simultaneous measurements by intact IRMA and PTH (1 84)-IRMA in end-stage renal disease patients have documented indistinguishable set-points for calcium-mediated PTH release but substantially higher values in the intact IRMA, consistent with the presence of PTH (7 84) fragments. The newer IRMA investigated in this study used a detection antibody that recognizes PTH (1 34) but not PTH (2 34), PTH (3 34), PTH (4 34), and PTH (5 34) (13). Thus, the first amino acid is required for detection, a finding consistent with the conclusion that this assay measures only the entire molecule. Because extremely distal amino-terminal amino acids are necessary for adenyl cyclase activation, this assay may also be viewed as a bioassay with regard to those functions of PTH that use camp. An assay that discriminates between PTH and large carboxy-terminal fragments is clearly advantageous in the management of secondary hyperparathyroidism. However, it was not obvious that such an assay would increase diagnostic sensitivity in primary hyperparathyroidism, a disorder not associated with increased rates of release and retention of hormone fragments. Using previously available assays, in vitro data suggest that intraglandular metabolism of PTH increases in states of hypercalcemia when hormone release rates are lower. Intraglandular metabolism decreases under hypocalcemic stimuli when secretion rates of active hormone increase (24, 25). Studies in hyperplastic parathyroid tissue from patients with secondary hyperparathyroidism demonstrate an increased proportion of carboxy-terminal fragments of PTH under high-calcium conditions (26). Animal and human studies confirm the secretion of variable amounts of carboxyterminal fragments under situations that stimulate or suppress PTH release (27, 28). These data support the idea that intraglandular degradation of PTH is dependent on ambient calcium concentration. Thus, hypercalcemia might be expected to be associated with an increase in relative amounts of PTH fragments as opposed to the whole molecule PTH (1 84). In primary hyperparathyroidism, this expectation does not seem to be true. Using older assays, Brossard et al. (29) compared the response to calcium infusions in normal individuals and primary hyperparathyroid patients with regard to proportions of intact PTH, carboxy-terminal PTH, and midcarboxyterminal PTH levels. Despite assay limitations, they concluded that carboxy-terminal fragments were not preferentially secreted in primary hyperparathyroidism. In recent years, the notion that fragments of PTH, such as PTH (7 84), were merely degradation products with no physiological role has been questioned (15, 30 32). In 1995, Inomata et al. (30) described a unique PTH receptor that binds PTH (1 84) and various carboxy-terminal fragments but not PTH (1 34). Nguyen-Yamamoto et al. (31) subsequently showed that carboxy-terminal fragments, particularly PTH (7 84), interact with a non-pth/pth-related protein receptor and can decrease calcium concentration in vivo in thyroparathyroidectomized rats. Animal data from Faugere et al. (32) also demonstrate that PTH (7 84) antagonizes both the calcemic effects of PTH (1 84) and its effects on bone dynamics. In uremic bone disease, for example, these fragments have been hypothesized to offer an explanation for the dissociation between measured PTH levels and observed skeletal effects (15). This study provides the first evidence that both PTH (1 84) and large carboxy-terminal fragments of PTH are produced in the parathyroid adenomas of patients with primary hyperparathyroidism. In only one previous study has parathyroid gland intracellular PTH been assessed for relative measurement of whole PTH and PTH fragment(s). Slatopolsky et al. (15) reported on the hyperplastic glands from six uremic patients, in whom 42% of intracellular PTH was non- PTH (1 84) and presumed to be similar to synthetic PTH (7 84). Thus, intraglandular production of truncated PTH fragments has now been confirmed in both primary and secondary hyperparathyroidism. It is possible that differences in protein, fat, and soft tissue content of the surgical specimen could have affected the measurement of intraglandular PTH (1 84) and intact PTH. However, the relative proportions of PTH (1 84) and car-

5 Silverberg et al. PTH(1 84) in Primary Hyperparathyroidism J Clin Endocrinol Metab, October 2003, 88(10): boxy-terminal PTH fragment in each parathyroid adenomas is unaffected by these factors. This report finds that both whole hormone and fragment are present in parathyroid adenomas and that their proportions are similar to those in the serum of affected patients. This is consistent with the hypothesis that in primary hyperparathyroidism, peripheral metabolism of hormone and fragment (in the circulation, liver, or kidney) does not alter the proportion of bioactive hormone. This would support a major role for the adenomatous parathyroid gland in the control of the molecular forms of PTH in this disorder. Further support for this role comes from the remarkable uniformity (lack of variability) seen in the percentage of PTH (1 84) in the circulation of adenoma patients. In normal individuals and groups of patients with secondary hyperparathyroidism, the proportion of PTH (1 84) ranges in a nearly Gaussian distribution from 20 to 90% (13). These patients with parathyroid adenomas had more than 60% bioactive PTH. Perhaps the preferential secretion of PTH (1 84) in this disease explains the distinct clinical profile that is seen despite very modest elevations of PTH. The demonstration of intraglandular production of PTH fragments in primary hyperparathyroidism raises some interesting and important questions. An increasing body of data supports potentially important physiological functions for fragments previously considered to be mere hormone degradation products (33, 34). The role of these molecular forms in primary hyperparathyroidism awaits elucidation. In summary, data from this study show that the PTH (1 84)-IRMA provides a significant improvement over previously available assays for PTH measurement in primary hyperparathyroidism. Although others have reported the intact assay to have sensitivities as high as 86% (35), the PTH (1 84)-IRMA correctly detects elevated PTH levels in nearly all patients, a significant improvement on the sensitivity of earlier PTH assays in this disease. It should be noted that the normal PTH levels measured in the older assays are inappropriately high in the face of hypercalcemia when any nonsuppressed level of PTH is physiologically inappropriate. Thus, the values obtained in the earlier assays were consistent with the diagnosis of primary hyperparathyroidism. However, many physicians are unwilling to make the diagnosis of primary hyperparathyroidism with seemingly normal PTH assay results. The introduction of this new assay may prevent the significant financial and psychological burden currently shouldered by many patients with hypercalcemia and normal PTH levels, who often undergo extensive diagnostic evaluation for alternative causes of hypercalcemia, particularly malignancy. It will allow the correct diagnosis of primary hyperparathyroidism in the vast majority of patients. Finally, data obtained using this assay may lead to important pathophysiological observations, which in turn may further our understanding of primary hyperparathyroidism. Acknowledgments We thank Dr. Leonard J. Deftos for help with assay performance and his helpful comments on the manuscript. Received August 12, Accepted June 26, Address all correspondence and requests for reprints to: Shonni J. Silverberg, M.D., Department of Medicine, College of Physicians and Surgeons, 630 West 168th Street, New York, New York sjs5@columbia.edu. This work was supported in part by National Institutes of Health Grant NIDDK References 1. Nussbaum SR, Potts JT 1994 Advances in immunoassays for PTH: clinical applications to skeletal disorders of bone and mineral metabolism In: Bilezikian JP, Marcus R, Levine MA, eds. The parathyroids. New York: Raven Press; Berson SA, Yalow RS, Aurbach GD, Potts JT 1963 Immunoassay of bovine and human parathyroid hormone. Proc Natl Acad Sci USA 49: Potts JT, Segre GV, Endres DB 1983 Current clinical concept: assessment of parathyroid function with an N-terminal specific radioimmunoassay for intact parathyroid hormone. Los Angeles: Nichols Institute; Nussbaum SR, Zahradnik RJ, Lavigne JR, Brennan GL, Nozawa-Ung K, Kim LY, Keutmann HT, Wang CA, Potts Jr JT, Segre GV 1987 Highly sensitive two-site immunoradiometric assay of parathyrin and its clinical utility in evaluating patients with hypercalcaemia. Clin Chem 33: Deftos LJ 2001 Immunoassays for PTH and PTHrP: clinical applications. In: Bilezikian JP, Marcus R, Levine MA, eds. The parathyroids. 2nd ed. New York: Academic Press; Martin KJ, Hruska KA, Freitag JJ, Klahr S, Slatopolsky E 1979 The peripheral metabolism of parathyroid hormone. N Engl J Med 302: Lepage R, Roy L, Brossard JH, Rousseau L, Dorais C, Lazure C, D Amour P 1998 A non-(1 84) circulating parathyroid hormone (PTH) fragment interferes significantly with intact PTH commercial assay measurements in uremic samples. Clin Chem 44: John MR, Goodman WG, Gao P, Cantor T, Salusky IB, Jueppiner H 1999 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 84: Kronenberg HM, Bringhurst R, Segre GV, Potts JT 2001 Parathyroid hormone biosynthesis and metabolism. In: Bilezikian JP, Marcus R, Levine MA, eds. The parathyroids. 2nd ed. New York: Academic Press; Bringhurst FR, Segre GV, Lampman GW, Potts Jr JT 1982 Metabolism of parathyroid hormone by Kupffer cells: analysis by reverse-phase highperformance liquid chromatography. Biochemistry 21: Dambacher MA, Fischer JA, Hunzier WH, Born W, Moran J, Roth HR, Delvin EE, Glorieux FH 1979 Distribution of circulating immunoreactive components of parathyroid hormone in normal subjects and in patients with primary and secondary hyperparathyroidism: the role of the kidney and of serum calcium concentration. Clin Sci 57: Segre GV, D Amour P, Hultman A, Potts Jr JT 1981 Effects of hepatectomy, nephrectomy, and nephrectomy, uremia on the metabolism of parathyroid hormone in the rat. J Clin Invest 67: Gao P, Scheibel S, D Amour P, John MR, Rao SD, Schmidt-Gayk H, Cantor TL 2001 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 16: Quarles LD, Lobough B, Murphy G 1992 Intact parathyroid hormone overestimates the presence and severity of parathyroid-mediated osseous abnormalities in uremia. J Clin Endocrinol Metab 75: Slatopolsky E, Finch JL, Clay P, Martin D, Sicard G, Singer G, Gao P, Cantor T, Dusso A 2000 A novel mechanism for skeletal resistance in uremia. Kidney Int 58: Szollar SM, Martin EME, Sartoris DJ, Parthemore JG, Deftos LJ 1998 Bone mineral density and indices of bone metabolism in spinal cord injury. Am J Phys Med Rehabil 77: Gao P, Scheibel SJ, D Amour P, Cantor TL 1999 Measuring the biologically active or authentic whole PTH with a novel IRMA without cross-reaction to the PTH(7 84) fragment. J Bone Miner Res 14(Suppl 1):S Silverberg SJ, Shane E, Jacobs TP, Siris E, Bilezikian JP 1999 The natural history of treated and untreated asymptomatic primary hyperparathyroidism: a ten year prospective study. N Engl J Med 341: NIH Conference 1991 Diagnosis and management of asymptomatic primary hyperparathyroidism: consensus development conference statement. Ann Intern Med 114: Silverberg SJ, Shane E, de la Cruz L, Dempster DW, Feldman F, Seldin D, Jacobs TP, Siris ES, Cafferty M, Parisien MV, Lindsay R, Clemens TL, Bilezikian JP 1989 Skeletal disease in primary hyperparathyroidism. J Bone Miner Res 4: Togashi K, Takahashi N, Ando K, Tsukamoto Y, Marumo F 1990 Comparison of different parathyroid hormone radioimmunoassays in uremic patients with secondary hyperparathyroidism. Int J Artif Organs 13: McCarthy JT, Klee GG, Kao PC, Hodgson SF 1989 Serum bioactive parathyroid hormone in hemodialysis patients. J Clin Endocrinol Metab 68:

6 4730 J Clin Endocrinol Metab, October 2003, 88(10): Silverberg et al. PTH(1 84) in Primary Hyperparathyroidism 23. Brossard JH, Lepage R, Cardinal H, Roy L, Rousseau L, Dorais C, D Amour P 2000 Influence of glomerular filtration rate on non-(1 84) parathyroid hormone (PTH) detected by intact PTH assays. Clin Chem 46: Kubler N, Krause U, Wagner PK, Beyer J, Rothmund M 1986 The secretion of parathyroid hormone and its fragments from dispersed cells of adenomatous parathyroid tissue at different calcium concentrations. Exp Clin Endocrinol 88: Mayer GP, Keaton JA, Hurst JG, Habener JF 1979 Effects of plasma calcium concentration on the relative proportion of hormones and carboxyl fragments in parathyroid venous blood. Endocrinology 104: Hanley DA, Ayer LM 1986 Calcium-dependent release of carboxyl-terminal fragments of parathyroid hormone by hyperplastic human parathyroid tissue in vitro. J Clin Endocrinol Metab 63: D Amour P, Labelle F, Lecavalier L, Plourde V, Harvey D 1986 Influence of serum Ca concentration on circulating molecular forms of PTH in three species. Am J Physiol 251:E680 E D Amour P, Palardy J, Bashali G, Malette LE, Delean A, Lepage R 1992 Modulation of circulating parathyroid hormone immunoheterogeneity in man by iodized calcium concentration. J Clin Endocrinol Metab 75: Brossard JH, Whitton S, Lepage R, D Amour P 1993 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 77: Inomata N, Akiyama M, Kubota N, Juppner H 1995 Characterization of a novel parathyroid hormone (PTH) receptor with specificity for the carboxylterminal region of PTH(1 84). Endocrinology 136: Nguyen-Yamamoto L, Rousseau L, Brossard JH, Lepage R, D Amour P 2001 Synthetic carboxyl-terminal fragments of parathyroid hormone (PTH) decrease ionized calcium concentration in rats by acting on a receptor different from the PTH/PTH-related peptide receptor. Endocrinology 142: Faugere MC, Langub MC, Malluche HH 2001 PTH (7 84) antagonizes the effects of PTH (1 84) on bone turnover in rats. J Bone Miner Res 16(Suppl 1):S Divieti P, Inomata N, Chapin K, Singh R, Juppner H, Bringhurst FR 2001 Receptors for the carboxy-terminal region of PTH)1 84) are highly expressed in osteocytic cells. Endocrinology 142: Divieti P, John MR, Juppner H, Bringhurst FR 2002 Human PTH(1 84) inhibits bone resorption in vitro via actions independent of the type 1 PTH/ PTHrP receptor. Endocrinology 143: Kao PC, van Heerden JA, Grant CS, Klee GG, Khosla S 1992 Clinical performance of PTH immunoradiometric assays. Mayo Clin Proc 67: