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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-1628
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Right arrow Calcium and Bone Metabolism
The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 1 283-289
Copyright © 2006 by The Endocrine Society

Acute Regulation of Circulating Parathyroid Hormone (PTH) Molecular Forms by Calcium: Utility of PTH Fragments/PTH(1–84) Ratios Derived from Three Generations of PTH Assays

Pierre D’Amour, Agnès Räkel, Jean-Hugues Brossard, Louise Rousseau, Caroline Albert and Tom Cantor

Centre de Recherche, Centre Hospitalier de l’Université de Montréal–Hôpital Saint-Luc and Department of Medicine (P.D., A.R., J.-H.B., L.R., C.A.), Université de Montréal, Montréal, Québec, Canada H2X 1P1; and Scantibodies Laboratory, Inc. (T.C.), Santee, California 92071

Address all correspondence and requests for reprints to: Pierre D’Amour, M.D., Centre de Recherche, Centre Hospitalier de l’Université de Montréal–Hôpital Saint-Luc, 264, Boulevard René-Lévesque est, Montréal (Québec), Canada H2X 1P1. E-mail: rechcalcium.chum{at}ssss.gouv.qc.ca.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: The quantitative evaluation of circulating PTH peaks revealed by PTH assays after HPLC separation constitutes the best way to study the behavior of PTH molecular forms, but it is also impractical.

Objective: The objective of the study was to investigate the regulation of circulating PTH molecular forms by calcium through the use of PTH fragments/PTH (1–84) ratios derived from PTH assays with different specificities before and after HPLC separation of circulating PTH.

Design: CaCl2 and Na citrate were infused in eight volunteers. PTH was measured in serum and HPLC fractions at different calcium concentrations in PTH assays reacting with regions 1–2 (CA), 12–18 (T), and 65–69 (C) of the PTH structure.

Results: From hypo- to hypercalcemia, the C/CA ratio had the highest range (1.92 to 9.75; P < 0.001), and the C/T ratio had a higher range (1.69 to 6.11; P < 0.01) than the T/CA ratio (1.15 to 1.86). Human (h) PTH (1–84) represented 32.7 and 4.3% of circulating PTH in hypo- and hypercalcemic HPLC profiles, respectively. These numbers were 5 and 0.9% for amino-terminal (N)-PTH, an amino-terminal form of PTH distinct from hPTH (1–84), 7.3 and 6.8% for non-(1–84) PTH or large C-PTH fragments with a partially preserved N structure, and 54.9 and 88.1% for C-PTH fragments missing a N structure. The HPLC C-PTH fragments to hPTH (1–84) ratio had the most extensive range (1.67 to 20.58). Despite their quantitative differences, all ratios identified identical behavior of PTH fragments relative to PTH (1–84).

Conclusions: PTH assay ratios are an adequate tool to investigate the modulation of PTH molecular forms, even if all PTH assays show some undesirable cross-reactivity with certain circulating forms of PTH.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
CARBOXYL-TERMINAL (C) PTH RIA (first generation) and gel chromatography were first used to separate circulating PTH(1–84), the biologically active form of the hormone on the type I PTH/PTHrP receptor, from inactive C-PTH fragments and to define the relationship between calcium concentration and circulating PTH molecular forms in normal individuals (1). Hypocalcemia, while stimulating PTH secretion, favored a low C-PTH fragments to PTH(1–84) ratio, whereas hypercalcemia did the reverse (1). When so-called intact PTH assays (second generation) became available, we were able to confirm these findings in a similar population by using the combination of a first- and second-generation PTH assay. We demonstrated high PTH levels with low mid- or C-PTH to intact PTH ratio values in hypocalcemia and low PTH levels with high mid- or C-PTH to intact PTH ratio values in hypercalcemia (2). Since these early studies, the world of circulating PTH molecular forms has become more complex, with the addition of non-(1–84) PTH fragments or large C-PTH fragments with a partially preserved amino-terminal (N) structure detected by intact PTH assays (3, 4, 5). Non-(1–84) PTH fragments represent approximately 20% of intact PTH after HPLC separation in a normal individual but up to 50% in renal failure patients because these are normally cleared by the kidney (3, 6, 7). The development of a third generation of PTH assays [whole PTH, cyclase-activating (CA) PTH, biointact PTH], based on epitopes that have required more or less of the first three amino acids of the PTH structure for antibody binding, has permitted elimination of non-(1–84) PTH fragments from the PTH assay, leaving only human (h) PTH(1–84) to be detected. This was soon discovered not to be the case, and a new N form of PTH, different from PTH(1–84), poorly reactive in intact PTH assays with a (12–18) epitope (8), possibly because of a posttranslational modification in that region (8), was identified. N-PTH represents 8% of CA-PTH in normal individuals but 15% in renal failure patients (8). The acute influence of calcium concentration on all these PTH molecular forms is yet to be investigated in normal individuals. Accordingly we performed a classical parathyroid function study in eight normal individuals, using three generations of PTH assays to identify all known PTH molecular forms separated by HPLC under various calcemic conditions.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Eight subjects, four men and four women, with a mean age of 36.4 ± 7.9 yr (range 25 to 44), participated in this study. They had no known diseases; were not taking any medication; and had normal values of ionized calcium, total calcium, phosphate, creatinine, alkaline phosphatase, and PTH.

Experimental protocol

The experimental protocol was approved by the research ethics committee of our research center, and all participants gave their signed, informed consent. Each subject was tested on two different occasions. Having consumed only water since the preceding evening, each subject was seated in an armchair, and catheters were placed in both antecubital veins for infusion and blood sampling. On the first occasion, serum Ca was increased for a 2-h period by means of an iv infusion of CaCl2 in 50 g/liter dextrose, which provided 125 µmol elemental Ca/kg·h. Four to 7 d later, serum Ca was decreased for 2 h by an iv infusion of Na citrate, which provided 29 µmol/kg·h. Procaine HCl (12.5 µmol/kg·h) was added to the Na citrate solution to relieve arm pain during the infusion. Blood samples were collected at 15-min intervals during both infusions. Ionized Ca was measured immediately and serum was stored at –75 C until PTH assays and HLPC fractionation of circulating PTH were performed.

Experimental methods

Ionized calcium (Ca2+) was measured by a Ca2+-specific electrode (Rapid Lab, model 348; Bayer Diagnostics, Toronto, Ontario, Canada). PTH was quantified in serum and HPLC fractions with three different PTH assays. The first, a third-generation PTH assay (Scantibodies Laboratory, Inc., Santee, CA) called CA-PTH or whole PTH, has been characterized extensively, reacts with hPTH(1–84) but not with hPTH(7–84) (9, 10), and has an epitope in the region (1–2) of the PTH structure in which removal of position 1 impairs most of the immunoreactivity (9). This assay recognizes both hPTH(1–84) and an N form of PTH distinct from hPTH(1–84) in the circulation (8). The second PTH assay, total (T) PTH, is a second-generation PTH assay from the same company. It is similar to intact PTH IRMA (Nichols Institute, San Juan Capistrano, CA) on head-to-head comparison (4), has a (12–18) epitope (4), reacts equally well with hPTH(1–84) and hPTH(7–84) (4), and recognizes hPTH(1–84) and non-(1–84) PTH fragments present in the circulation but reacts poorly with N-PTH, possibly because of a posttranslational modification in the epitope region (4). Finally, the last assay is an in-house C RIA, which uses [tyr(53)] hPTH(53–84) as tracer and hPTH(39–84) as standard. The assay reacts two to three times better on a molar basis with hPTH(39–84) than with hPTH(1–84) and reacts with the region (65–69) of the PTH structure. It is mainly a C-PTH fragment assay (1, 2, 3).

HPLC analysis

Circulating PTH molecular forms from a pool of all eight sera at a given calcium concentration were extracted with Sep-Pak Plus C-18 cartridges (Waters, Milford, MA), as described by Bennett et al. (11). One C-18 cartridge was designated for each 3 ml of serum or medium. Samples were eluted from the cartridge with 3 ml of 800 ml/liter acetonitrile in 1 g/liter 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/liter trifluroacetic acid for HPLC analysis. Each 2-ml sample was loaded on a Waters C18 µBondapak analytical column [300 x 3.9 mm (inner diameter)] and eluted with a noncontinuous linear gradient of acetonitrile in 1 g/liter trifluoroacetic acid. The gradient ranged from 15–23% in 25 min, 23–30% in 5 min, and 30–33% in 30 min. The gradient was delivered at 1.5 ml/min with a solvent delivery system (Agilent Technologies, Wilmington, DE). The 1.5-ml fractions were evaporated, freeze dried, and reconstituted to 1 ml with 7 g/liter BSA in water; adequate volumes were then measured in the various PTH assays. Control experiments were performed with hPTH(1–84) added to hypoparathyroid serum to ensure that PTH degradation did not occur during the various procedures. After HPLC separation, a single peak of immunoreactivity coeluting with hPTH(1–84) was detected by the three PTH assays. Immunoreactive PTH recovery, through all of these procedures, was calculated by comparing the original serum PTH value with the sum of PTH immunoreactivity across all HPLC fractions.

Statistical analysis

The results are expressed as means ± SD. The functional status of the parathyroid glands of each individual was analyzed using a logistic model corresponding to the four-parameter equation:

where Y = serum PTH concentration; x = ionized calcium concentration; A = maximal PTH or ratio response during hypocalcemic stimulation; b = slope of the mathematical function at the set point, which reflects the sensitivity to Ca2+ change; C = the set point of serum Ca2+ concentration or Ca2+ value corresponding to 50% change in PTH or ratio value; D = the nonsuppressible fraction of serum PTH or ratio value at maximal hypercalcemic inhibition (1, 2, 3). All points, derived from combined CaCl2 and Na citrate studies, were used for each analysis. Fitting of the calculated curve to the experimental points was evaluated via the square of the correlation coefficient (R2). C-PTH fragments to PTH(1–84) ratios, which reflect mainly the behavior of C-PTH fragments, and PTH(1–84) to C-PTH fragment ratios, which reflect mainly the behavior of PTH(1–84), were also analyzed using the same logistic model because these ratios’ relationship to ionized calcium could be fitted by a sigmoidal curve. Differences between assays or ratios were assessed by a one-way repeated-measures ANOVA followed by the Student-Newman-Keuls multiple comparison test for 2 x 2 comparison. Differences between PTH assays and PTH ratios set points were assessed by a two-way repeated-measures ANOVA (two factor repetition) followed by the same multiple comparison test. HPLC profiles were examined by planimetry, evaluating the area under each peak in each assay. For the final quantitative HPLC results, hPTH(1–84) values came from the mean of CA and T-PTH assays, N-PTH values from the CA-PTH assay, non-(1–84) PTH values from the T PTH assay, and C-PTH fragments values from the C-PTH assay.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
The results of parathyroid function analysis with each of the three PTH assays and PTH ratio analysis with the same assays are outlined in Table 1Go. Figure 1Go also illustrates how these results were achieved in a representative, normal individual through the analysis of all experimental points. Both the stimulated and nonsuppressible fractions of PTH were highest in the C-PTH assay (P < 0.001) and higher in the T-PTH assay (P < 0.01) than the CA-PTH assay. The negative slope of the sigmoidal function, the set point of PTH stimulation by calcium, and the fitting of the data to the function were similar for all three PTH assays. When the PTH ratios were obtained with PTH assays, with C-PTH fragments as numerator and predominantly hPTH(1–84) as denominator, both the minimum value in hypocalcemia and the maximum value in hypercalcemia were highest for the C-PTH to CA-PTH ratio (P < 0.05 to < 0.001), and higher for the C-PTH to T-PTH ratio (P < 0.001) than the T-PTH to CA-PTH ratio. The positive slope of the sigmoidal function, the set point of PTH molecular form modulation by calcium, and the fitting of the data were similar for all three ratios. On the other hand, when ratios were obtained with PTH assays with hPTH(1–84) as numerator and C-PTH fragments as denominator, the slope of the sigmoidal function remained negative. The maximum value in hypocalcemia and the minimum value in hypercalcemia were highest for the CA-PTH to T-PTH ratio (P < 0.001) and higher for the T-PTH to C-PTH ratio (P < 0.01) than for the CA-PTH to C-PTH ratio. Again slope, set point, and fitting did not differ among the three ratios. The set points of PTH ratio modulation by calcium (Table 1Go) were significantly higher than the set points of PTH stimulation by calcium (P < 0.05 to P < 0.001).


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  		TABLE 1. Analysis of parathyroid function and PTH assay ratios

 


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  		FIG. 1. Analysis of parathyroid function and PTH assay ratios in a representative normal individual with all experimental points obtained during CaCl2 and Na citrate infusions. A four-parameter sigmoidal curve mathematical model was used to analyze the data as described in Subjects and Methods.

 
Figure 2Go illustrates the relationship between the various PTH assays according to all the experimental points from the eight subjects. The CA-PTH results were highly correlated to the T-PTH results (r2 = 0.988). The C-PTH results were also correlated to the CA-PTH (r2 = 0.8333) and T-PTH (r2 = 0.850) results.



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  		FIG. 2. Analysis of the relationship between PTH results obtained with the three PTH assays using all experimental points from the eight subjects obtained during CaCl2 and Na citrate infusions.

 
We next tried to evaluate for each individual the calcium concentration at which most of the changes in PTH ratios had already occurred (Table 2Go). Basal PTH levels and PTH ratios were set at a calcium concentration of 1.22 ± 0.02 mmol/liter. When decreasing serum calcium, already at 1.17 ± 0.07 mmol/liter, just below the lower limit of normal, stimulated PTH levels were already at their maximum, and the PTH ratios had achieved definitive values. The latter were all significantly different from values obtained in normocalcemia (P < 0.01 to P < 0.001), except those obtained with the CA-PTH to T-PTH ratio that did not differ from baseline. When increasing serum calcium, already at 1.32 ± 0.07 mmol/liter, at the upper limit of normal values, most of the decrease in PTH levels and changes in PTH ratios had already happened. All the results obtained at 1.32 ± 0.07 mmol/liter of calcium were significantly different from those at baseline (P < 0.05 to < 0.001) except for those obtained with the T-PTH to CA-PTH ratio, which did not differ from baseline. Most of these ratio results did not differ between 1.32 ± 0.07 and 1.69 ± 0.05 mmol/liter of calcium, even if suppressible PTH values measured with the three assays were slightly different.


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  		TABLE 2. Changes in PTH levels and PTH ratios in relation to Ca2+ concentrations

 
Figure 3Go illustrates the HPLC profile of circulating PTH molecular forms analyzed with the three PTH assays at Ca2+ concentrations very near those of Table 2Go. Planimetric evaluation of the HPLC peaks is summarized in Table 3Go. Four regions of interest corresponding to hPTH(1–84) (region 49–52), N-PTH (region 45–58), non-(1–84) PTH (region 35–42), and C-PTH fragments (region 10–33) were identified. hPTH(1–84) was identified by all three PTH assays, N-PTH mainly by the CA-PTH assay, non-(1–84) PTH fragments mainly by the T-PTH assay, and C-PTH fragments exclusively by the C-PTH assay. The dynamic range of hPHT(1–84) values went from 0.24 to 6.51 pmol/liter from hyper- to hypocalcemia with a basal value of 2.04 pmol/liter. This is very close to what is observed with the CA-PTH assay (Table 1Go), the slight difference corresponding to the detection of N-PTH. The latter represented 0.05–0.93 pmol/liter of CA-PTH, going from hyper- to hypocalcemia and was less well detected by the T-PTH assay. Non-(1–84)PTH fragments were best detected by the T-PTH assay with a dynamic range of 0.38–1.35 pmol/liter from hypercalcemia to hypocalcemia, representing 6.8% of total PTH in the former situation and approximately 7.3% in the latter. Finally, in all circumstances, C-PTH fragments represented the bulk of circulating PTH with a dynamic range going from 4.94 to 10.13 pmol/liter from hyper- to hypocalcemia, representing 88% of circulating PTH in the former situation and 55% in the latter.



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  		FIG. 3. HPLC profiles of circulating PTH under various calcemic conditions. At each calcium concentration, a pool of serum, coming from the eight experimental subjects, was processed as described in Subjects and Methods, and the HPLC fractions were analyzed with the three PTH assays. Planimetric evaluation of the various regions of interest is presented in Table 3Go.

 

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  		TABLE 3. Concentrations of PTH molecular forms derived from HPLC profiles analyzed with three different PTH assays

 
PTH ratios derived from HPLC results disclose a dynamic range of 0.154–0.208 for the N-PTH to hPTH(1–84) ratio, 0.257–1.58 for the non-(1–84) PTH to hPTH(1–84) ratio, and of 1.67–20.58 for the C-PTH to hPTH(1–84) ratio.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The aim of this study was to evaluate the behavior of all known circulating PTH molecular forms as a function of calcium concentration in normal individuals. To accomplish this, we tested classical CaCl2 and Na citrate infusions in normal individuals, as we have done previously (1, 2, 3), with three PTH assays, which reacted differently with circulating PTH molecular forms but covered all of them (1, 2, 3, 4), and HPLC separation of circulating PTH (3, 4). We wanted to see whether ratios of the PTH assays, which detected predominantly hPTH(1–84) and/or C-PTH fragments, could predict the behavior of circulating PTH molecular forms as a function of Ca2+, using HPLC separation of these molecular forms as the gold standard.

There was excellent relationship between PTH results obtained with the three PTH assays. This was particularly evident for the CA and T-PTH assays with a correlation coefficient of almost 1. Quantitative analysis of parathyroid function reflected the capacity of each assay to recognize more or less of the particular molecular forms of PTH. Because the C-PTH assay detects fragments that make up the bulk of circulating PTH (1, 2, 3) and accumulate relative to hPTH(1–84) in hypercalcemia (1, 2, 3), it is not surprising that it produced the highest stimulated and nonsuppressible PTH values. Because the T-PTH assay detects non-(1–84) PTH fragments that are not seen by the CA-PTH assay, it is also expected to have higher values than the CA-PTH assay, even if the latter detects N-PTH better than the T-PTH assay (4). On the other hand, sensitivity to Ca2+ changes (slope), the set point of PTH stimulation by Ca2+, and the fitting of the data to the sigmoidal curve did differ among the three PTH assays.

To evaluate the behavior of circulating PTH molecular forms, we studied various PTH assay ratios. The C-PTH assay detects mainly C-PTH fragments, the T-PTH assay, non-(1–84) PTH fragments and hPTH(1–84) as well, and the CA-PTH assay, mostly hPTH(1–84) but also N-PTH (1, 2, 3, 4). Because the immunoreactivity of circulating PTH molecular forms may vary in relation to the PTH standard used in each assay, quantitative ratio results should be considered an approximation. All C-PTH fragments to hPTH(1–84) ratios, including the C-PTH to CA-PTH, the C-PTH to T-PTH, and the T-PTH to CA-PTH ratios, behave similarly with low values in hypocalcemia and high values in hypercalcemia, reflecting the dominant role of hPTH(1–84) in the former situation and C-PTH fragments and non-(1–84) PTH fragments in the latter (3). These ratios had different absolute values, which reflected the capacity of each assay to interact with more or less C-PTH fragments, hPTH(1–84) being also contaminated by non-(1–84) PTH fragments in the T-PTH assay and N-PTH in the CA-PTH assay. The behavior of these ratios differed from the parathyroid function sigmoidal curves by having a positive slope and a much higher set point. This indicates that the mechanisms that lead to the formation and secretion of C-PTH fragments are mainly activated when higher Ca concentrations are observed in the circulation (3). Even when inverse ratios such as CA-PTH to C-PTH, T-PTH to C-PTH, and CA-PTH to T-PTH ratios were obtained, a similar situation prevailed with a higher set point of Ca2+ stimulation but with much lower ratio values. These ratios had a negative slope, higher values in hypocalcemia, and lower values in hypercalcemia, but the values were always less than 1, as expected from the lower concentration of hPTH(1–84) at all calcium concentrations.

We next looked at PTH and ratio values at various Ca2+ concentrations to establish the earliest Ca2+ concentration at which most changes in PTH values and ratios had already occurred. These calcium values were 1.17 ± 0.07 mmol/liter in hypocalcemia and 1.32 ± 0.070 mmol/liter in hypercalcemia. This corresponds, in fact, to the lower and upper limits of the normal Ca2+ range (1.19–1.34 mmol/liter), indicating that most modifications in PTH and PTH ratio values belong to normal physiology. Getting more hypocalcemic or hypercalcemic added very little to what had been already observed within the normal range of Ca2+ values.

With HPLC separation of circulating PTH molecular forms, we were able to quantitate hPTH(1–84), N-PTH, non-(1–84) PTH, and C-PTH fragments to more exact values and generate ratios of each molecular form to hPTH(1–84). C-PTH fragments were the dominant form of circulating PTH under all calcemic conditions, followed by hPTH(1–84), non-(1–84) PTH and, finally N-PTH. The behavior of C-PTH fragments, non-(1–84) PTH, was similar when compared with hPTH(1–84), with the two ratios reaching their highest value in hypercalcemia and their lowest in hypocalcemia, suggesting similar behavior and possible origin for C-PTH fragments. N-PTH, on the other hand, was present in circulation at a relatively constant fraction of hPTH(1–84), suggesting a different behavior from C-PTH fragments or non-(1–84) PTH.

One must add another dimension to these results before drawing any conclusions. It is uncertain whether all non-(1–84) PTH fragments and all C-PTH fragments have an intact C-terminal structure including position 84 (5). hPTH(7–84) has been used experimentally (12, 13, 14, 15) to study the biological effects of non-(1–84) PTH fragments before it was known to be the main secreted non-(1–84) PTH fragments (5), hPTH(7–84) and, to a lesser extent, C-PTH fragments have biological effects opposite to hPTH(1–84) in vivo (12, 13, 14) and in vitro (15, 16) by acting on a different C-PTH receptor. hPTH(7–84) is probably 10 times more biologically active on a molar basis than C-PTH fragments (13, 15, 16), the prototype of which is hPTH(39–84). Nonetheless, C-PTH fragments represent 90% of circulating C-PTH fragments, whereas hPTH(7–84) and equivalent molecules are only 10%, even in renal failure. This means that on a molar basis hPTH(7–84) and C-PTH fragments probably could be equivalent biologically (13). Ratios that favored C-PTH fragments, like the C-PTH to CA-PTH or C-PTH to T-PTH ratios, even if they have relatively higher values, may not be more important than the T-PTH to CA-PTH ratio with much lower values.

Overall, this study demonstrates that the use of PTH assay ratios can help to investigate the behavior of circulating PTH molecular forms and that this is true even if what is measured is contaminated in part by hPTH(1–84) for C-PTH or non-(1–84) PTH for T-PTH and N-PTH for CA-PTH. Ratios obtained with the CA and T-PTH assays also behave identically. These results are minimally improved after HPLC separation of circulating PTH molecular forms, but even this does not change the interpretation of PTH molecular form regulation by calcium. This study validates the use of PTH ratios obtained by PTH assays with different specificities to study the behavior of PTH molecular forms in experimental and clinical studies as long as such assays have been appropriately characterized to identify their epitopes and their cross-reactivity with circulating PTH molecular forms (3, 4, 5).


   Acknowledgments
 
The authors thank Manon Livernois for typing this manuscript and Ovid Da Silva for editing it.


   Footnotes
 
This work was supported by Grant MOP-7643 from the Canadian Institutes of Health Research.

First Published Online October 11, 2005

Abbreviations: CA, Cyclase-activating; Ca2+, ionized calcium; h, human; T, total.

Received July 21, 2005.

Accepted October 4, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. 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–E687
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