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Endocrinology and Metabolism |
1 Centre de Recherche, Centre Hospitalier de l’Université de Montréal (CHUM), Hôpital Saint-Luc, and Departments of 2
Medicine and 3
Biochemistry, Hôpital Saint-Luc and Université de Montréal, Montréal, Québec, Canada.
4 Scantibodies Laboratory Inc., Santee, CA.
aAddress correspondence to this author at: Centre de Recherche, CHUM, Hôpital Saint-Luc, 264, boul. René-Lévesque est, Montréal, Québec, Canada H2X 1P1. Fax 514-412-7314; e-mail rechcalcium.chum{at}ssss.gouv.qc.ca.
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Abstract |
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Methods: Sera obtained from six healthy individuals and six patients with primary hyperparathyroidism, and six serum pools from dialysis patients with different PTH concentrations were fractionated by HPLC and analyzed by four different PTH assays. Each assay was characterized by saturation analysis of its detection antibody and capacity to react with different PTH fragments. Human PTH(1–84) [hPTH(1–84)] calibrators were normalized to an in-house hPTH(1–84) calibrator.
Results: The cyclase-activating PTH (CA-PTH) assay had an early (1, 2,) epitope and reacted only with hPTH(1–84). The other assays had epitopes in region (13–34). Total and intact PTH assays had epitopes proximal to position 18 and reacted equally well with hPTH(1–84) and hPTH(7–84), and the Elecsys PTH assay had an epitope distal to position 19, being saturable by hPTH(18–48) and also reacting with [Tyr34]hPTH(19–84). The HPLC profiles obtained with these assays showed that non-(1–84) PTH fragments did not react in the CA-PTH assay, as expected. The amount of non-(1–84) PTH detected by the other three assays was similar when the assay results were normalized to a common calibrator.
Conclusions: The results suggest that the amount of non-(1–84) PTH detected by epitopes proximal or distal to position 19 of the PTH structure is identical, indicating a common minimum structure starting before position 19. This in turn points to a probable high-affinity interaction with the C-PTH receptor, as observed previously with [Tyr34]hPTH(19–84) in various cell lines and in mouse osteocytes with PTH/PTHrP type I receptor ablation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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20% of I-PTH in healthy individuals and up to 50% in patients with terminal renal failure (dialysis) (2)(3). These fragments are different from the smaller C-PTH fragments, which make up the majority of circulating PTH and do not react in I-PTH assays (2). Like other C-PTH fragments, non-(1–84) PTH fragments are cleared by the kidney and accumulate in renal failure patients proportionally to the degree of renal failure (4)(5). They are both secreted by the parathyroid glands and generated during the peripheral metabolism of hPTH(1–84) (5). The exact N-terminal structure of non-(1–84) PTH fragments remains unknown. Human PTH(7–84) [hPTH(7–84)], a commercially available surrogate for all non-(1–84) PTH or N-truncated fragments, was used to demonstrate that such fragments react in I-PTH assays (2)(3). Furthermore, hPTH(7–84) has been found to exert specific biological activities of non-(1–84) PTH fragments in vivo and in vitro via a C-PTH receptor (6)(7)(8)(9). hPTH(7–84) is hypocalcemic in parathyroidectomized rats (6)(7) and antagonizes the calcemic effect of hPTH(1–84) and hPTH(1–34) in the same rat model (6)(7)(8). It is also a potent inhibitor of bone resorption induced by various substances in vitro (9), of osteoclast and osteoblast formation, and of bone turnover in thyroparathyroidectomized uremic rats (8). These studies have also shown that the biological effects of hPTH(7–84) are independent of the PTH/PTHrP type I receptor and of cAMP production and are exerted via a yet to be cloned C-PTH receptor (6)(7)(9).
To gain more information on the N-terminal structure of non-(1–84) PTH fragments, we took an immunologic approach, based on N-terminal epitopes found in commercial I-PTH assays, to ascertain a minimal structure for all non-(1–84) PTH fragments. Our results suggest that all non-(1–84) PTH fragments start before position 19 of the PTH structure.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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experimental protocol
Blood was obtained by venipuncture from healthy individuals and patients with primary hyperparathyroidism in the research laboratory and was centrifuged immediately. The serum was stored at –90 °C until further processing. Sera obtained from the biochemistry laboratory had been centrifuged within a maximum of 4 h from collection and stored at –90 °C until further processing. Indices of calcium and phosphorus metabolism and basal PTH concentrations were measured in serum either fresh or kept at –90 °C. The same serum was used for HPLC analysis. For renal failure, six pools were constituted with various PTH concentrations (6–90 pmol/L), by use of several sera. Although it is possible that pooling may have produced PTH molecular forms not representative of individual specimens, we have found in the past that this approach gives HPLC profiles similar to those obtained in single individuals (3).
experimental methods
The synthetic PTH peptides [Cys8]hPTH(1–8), hPTH(1–34), hPTH(2–38), hPTH(13–34), hPTH(18–48), hPTH(1–84), and hPTH(7–84) were purchased from BACHEM. Mutated [Tyr34]hPTH(19–84), [Tyr34]hPTH(24–84), and [Tyr34]hPTH(28–84) were generously provided by Dr. H. Jüppner (Massachusetts General Hospital, Boston, MA).
Total calcium, phosphate, alkaline phosphatase, and creatinine were measured by automated colorimetric methods. Total PTH (T-PTH) and cyclase-activating PTH (CA-PTH) were quantified by commercial assays provided by Scantibodies Laboratory Inc. The detection limit of the T-PTH assay is reported to be 0.13 pmol/L, and the intraassay CV is 4.8% at 2.15 pmol/L. For the CA-PTH assay, the detection limit is 0.1 pmol/L, and the intraassay CV is 6.2% at 3.2 pmol/L. I-PTH was measured with the Allegro Intact PTH assay from Nichols Institute. The detection limit is 0.1 pmol/L, and the intraassay CV is 3.4% at 4 pmol/L. The Elecsys PTH (E-PTH) was measured with the Roche Diagnostics assay. The detection limit is 0.127 pmol/L, and the intraassay CV is 5.4% at 3.18 pmol/L.
The nature of each assay epitope was studied through saturation analysis of the amino-terminal antibody with the synthetic PTH peptides [Cys8]hPTH(1–8), hPTH(1–34), hPTH(2–38), hPTH(13–34), and hPTH(18–48). Assay specificity was studied by use of various PTH fragment calibrators and through determination of the capacity of each assay to recognize circulating PTH molecular forms present in the various populations after HPLC separation. To confirm that differences obtained in PTH measurements within each population were not related to standardization differences, we calibrated each assay hPTH(1–84) calibrator against an in-house hPTH(1–84) calibrator, the concentration of which was determined by amino acid analysis. This relationship (slope) was used to calculate corrected PTH values, which in turn served to determine the amount of PTH present in each HPLC peak.
Circulating PTH molecular forms from all sera were extracted by use of Waters Sep-Pak Plus C-18 cartridges, as described by Bennett et al. (10). One C-18 cartridge was used for each 3 mL of serum. Up to 15 mL of serum was used for healthy individuals. Samples were eluted from the cartridge with 3 mL of 800 mL/L acetonitrile in 1 g/L trifluoroacetic acid. Acetonitrile was evaporated from the eluate with nitrogen, and the residual volume was freeze-dried and then reconstituted in 2 mL of 1 g/L trifluoroacetic acid for HPLC analysis. Each 2-mL sample was loaded on a Waters C18 µBondapak analytical column [300 x 3.9 mm (i.d.)] and eluted with a noncontinuous linear gradient of acetonitrile in 1 g/L trifluoroacetic acid. The gradient ranged from 15% to 23% in 25 min, 23% to 30% in 5 min, and 30% to 33% in 30 min. The gradient was delivered at 1.5 mL/min with a Hitachi Model L-6200 solvent delivery system. The 1.5-mL fractions were evaporated, freeze-dried, and reconstituted to 1 mL with 7 g/L bovine serum albumin in water; adequate volumes were then measured in the various PTH assays. Control experiments were performed with the hPTH(1–84) calibrator added to serum from patients with hypoparathyroidism to ensure that PTH degradation did not occur during the various procedures. A single peak of immunoreactivity coeluting with hPTH(1–84) in all eluates was detected by the four PTH assays. Immunoreactive PTH recovery by all PTH assays through all of these procedures was >77% (77–90%) for the 18 HPLC runs, based on comparison of the original serum PTH values with the sum of PTH immunoreactivity across all HPLC fractions.
data analysis
The results are expressed as the mean (SD). Differences between groups were analyzed by one-way ANOVA on log-transformed values, followed by the Student–Newman–Keuls multiple-comparisons test. Differences within groups were analyzed by ANOVA for repeated measures on log-transformed values, followed by the Student–Newman–Keuls multiple-comparisons test. HPLC profiles were evaluated planimetrically with Origin 4.1 software (Microcal Software Inc.).
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Results |
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. Saturation analysis of the detection antibody is shown on the left, and immunoreactivity with the different PTH calibrators is shown on the right. The detection antibody of the CA-PTH assay was saturable by hPTH(1–34) and [Cys8]hPTH(1–8), minimally saturable by hPTH(2–38), and not at all saturable by hPTH(13–34) and hPTH(18–48). This antibody reacted only with standard hPTH(1–84). The detection antibody in the T-PTH assay could be saturated by hPTH(1–34), hPTH(2–38), and hPTH(13–34), but not by [Cys8]hPTH(1–8) or hPTH(18–48). It reacted equally with hPTH(1–84) and hPTH(7–84). The detection antibody in the I-PTH assay behaved similarly to that of the T-PTH assay with the exception of a minor immunoreactivity of [Tyr34]hPTH(19–84) in the I-PTH assay. Finally, the E-PTH assay differed from the T-PTH and I-PTH assays in that it could be saturated by hPTH(18–48) and reacted slightly better with hPTH(1–84) than with hPTH(7–84) and [Tyr34]hPTH(19–84). It also reacted slightly with [Tyr34]hPTH(24–84) but not with [Tyr34]hPTH(28–84).
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The biochemical differences between groups are shown in Table 1
. Total calcium was highest in patients with primary hyperparathyroidism and higher in hemodialysis patients than in healthy individuals. Phosphate was lowest in the primary hyperparathyroidism group and highest in the renal failure group. Alkaline phosphatase was more increased in primary hyperparathyroidism than in hemodialysis patients, with concentrations in both groups being higher than in healthy individuals. PTH was higher in primary hyperparathyroidism patients than in healthy individuals and was highest in hemodialysis patients. Within each group, the CA-PTH assay gave the lowest PTH results. Higher results were obtained with the I-PTH and E-PTH assays in healthy individuals compared with the CA-PTH assay. The E-PTH assay gave the highest results for patients with primary hyperparathyroidism, whereas results with the T-PTH and I-PTH assays were higher than in healthy individuals. The results obtained with the T-PTH, I-PTH, and E-PTH assays were higher in hemodialysis patients than in healthy individuals but did not differ from each other. The mean (SD) CA-PTH/T-PTH ratio was 0.85 (0.11) in healthy individuals, 0.68 (0.08) in patients with primary hyperparathyroidism, and 0.70 (0.11) in dialysis patients. To ensure that between-assay differences were not related to standardization, we compared results from hPTH(1–84) calibrator for each assay along with an in-house hPTH(1–84) calibrator (Fig. 2
). The in-house calibrator gave values corresponding to 0.957 of CA-PTH values, 1.06 of T-PTH values, 0.918 of I-PTH values, and 0.814 of E-PTH values. When corrected for these standardization differences (Table 2
), CA-PTH values remained lower in the three populations, but there were no significant differences among the three other assays.
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The mean HPLC profiles obtained by each of the four PTH assays within each group are shown in Fig. 3
. Each profile represents the mean (SD) of six different HPLC runs. Table 2
provides a quantitative analysis of HPLC peaks applied to mean basal corrected PTH values. The CA-PTH assay reacted with two peaks of immunoreactivity: a major peak in position 40–41 that coeluted with the hPTH(1–84) calibrator and a minor, slightly less hydrophobic peak in position 34–35. The latter peak represented 10% of the immunoreactivity in healthy individuals, 7% in primary hyperparathyroidism, and 14% in renal failure patients on dialysis. Occasionally, two very minor peaks of immunoreactivity were also observed with the CA-PTH assay, in positions 29 and 25, in dialysis patients with the highest PTH concentrations. Three peaks of immunoreactivity were identified by the other PTH assays. The first peak, in position 40–41, corresponded to hPTH(1–84), the second peak, in position 34–35, to the second peak identified by the CA-PTH assay, and the third peak, in position 24–30, corresponded to non-(1–84) PTH or N-truncated PTH fragments. The latter peak represented 15–21% of the immunoreactivity in healthy individuals, 20–25% in patients with primary hyperparathyroidism, and 32–35% in dialysis patients.
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The amount of hPTH(1–84) (peak 1) identified in HPLC profiles by the four PTH assays was similar in healthy individuals and dialysis patients. In patients with primary hyperparathyroidism, the E-PTH assay measured a higher hPTH(1–84) concentration than the CA-PTH assay. The amount of modified hPTH(1–84) detected (peak 2) was generally higher in the CA-PTH and E-PTH assays than in the other two assays, particularly in healthy individuals and patients with primary hyperparathyroidism. The same tendency occurred in dialysis patients but did not reach statistical significance. Finally, the amount of non-(1–84) PTH detected by the T-PTH, I-PTH, and E-PTH assays was similar within each population, whereas the CA-PTH assay reacted poorly with this peak, as expected.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We had first to demonstrate that the selected commercial PTH assays indeed had different epitopes in region 13–34. We used a third-generation PTH assay as a control (11)(12). This assay reacts with the first two amino acids of the PTH structure and requires an intact position 1 (5). The other three PTH assays, although sold under various commercial names, had their main epitopes in region 13–34. The T-PTH assay had the most proximal epitope, could not be saturated with hPTH(18–48), and did not react with [Tyr34]hPTH(19–84). The I-PTH assay had a slightly more distal epitope because it reacted slightly with [Tyr34]hPTH(19–84). The E-PTH assay clearly had the most distal epitope, being totally saturated by hPTH(18–48) and reacting fully with [Tyr34]hPTH(19–84) and partially with [Tyr34]hPTH(24–84), but not at all with [Tyr34]hPTH(28–84). Combined, these assays permitted us to identify N-truncated fragments starting at position 2 and going up to position 24, with the T-PTH and I-PTH assays going up to position 19 and the E-PTH assay up to position 24, a difference of approximately five amino acids.
The data obtained in various populations with the different assays appeared to favor the hypothesis that most distal epitopes in the 13–34 PTH region give higher PTH results (Table 1
). The CA-PTH/T-PTH ratios observed in the three populations were also similar to our previously published results (12)(13), but when we corrected results for differences observed in the amount of hPTH(1–84) calibrator present in the assays, differences among the three PTH(13–34) assays disappeared. This indicates that standardization differences contribute to different results among commercial PTH assays. Furthermore, when we analyzed the HPLC profiles of circulating PTH molecular forms in six samples from each population, we were unable to demonstrate a real difference among the HPLC profiles obtained by the three PTH(13–34) assays. The amounts of non-(1–84) PTH or N-truncated fragments detected by each assay in the three populations were similar, suggesting that the N-structures of these fragments start before position 19. Other differences were nonetheless noted. The capacities of the T-PTH and I-PTH assays to react with a new amino-terminal molecular form of PTH (13) migrating in position 34–35 and best detected by the CA-PTH assay were limited compared with the E-PTH assay, which recognized this molecular form nearly as well as the CA-PTH assay. This is compatible with our original hypothesis that this PTH molecular form is posttranslationally modified in region 15–20, making it less reactive in assays that have their main epitope in that region. Another difference is that the E-PTH assay detected more immunoreactivity in the 1–84 region than the CA-PTH assay in patients with primary hyperparathyroidism. The exact reason for this remains unclear. We cannot eliminate the possibility that the hPTH(1–84) peak contains other molecular forms of PTH not separated adequately from hPTH(1–84) and reactive in the E-PTH assay or that the detection antibody in the E-PTH assay reacts slightly better with hPTH(1–84) (Fig. 1
).
Because the N-terminal structures of non-(1–84) PTH or N-truncated PTH fragments appear to start before position 19, we begin to speculate on their interaction with the C-PTH receptor. [Tyr34]hPTH(19–84) has been demonstrated to bind to the C-PTH receptor in various cell lines and to displace the tracer as well as hPTH(1–84) (14). The synthetic fragment hPTH(39–84), which is missing an N-terminal structure and can be considered representative of smaller circulating C-PTH fragments, was at least 10 times less potent on a molar basis in displacing the tracer from the receptor. Similar findings were reported in mouse osteocytes in which the PTH/PTHrP type I receptor had been deleted (15). hPTH(1–84), [Tyr34]hPTH(19–84), and hPTH(24–84) were equally efficient in displacing the tracer, whereas hPTH(39–84) was 20-fold less effective (15). However, because smaller C-PTH fragments represent 80% of circulating PTH whereas non-(1–84) PTH represents <10%, they could still have a significant biological impact (7)(9). Combined with our results, these observations indicate that circulating non-(1–84) PTH fragments are likely to be active on the C-PTH receptor and that data obtained with hPTH(7–84) are also likely to apply to circulating N-truncated PTH fragments in humans.
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Acknowledgments |
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Footnotes |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), [Tyr34]hPTH(19–84) (
), [Tyr34]hPTH(24–84) (
), and [Tyr34]hPTH(28–84) (
). The CA-PTH assay had an early epitope requiring an intact position 1 and reacted only with hPTH(1–84). The other three PTH assays had (13–34) epitopes before position 18 in the T-PTH and I-PTH assays and after position 19 in the E-PTH assay.
