A non-(1–84) circulating parathyroid hormone (PTH) fragment interferes significantly with intact PTH commercial assay measurements in uremic samples

¹ Centre de recherche clinique du CHUM, Pavillon Saint-Luc, Montréal, Quebec, H2X 1P1 Canada.

² Institut de Recherches Cliniques de Montréal and Départements de
³ Médecine et de
⁴ Biochimie, Université de Montréal, Montréal, Quebec, H3C 3J7 Canada.
^a Address correspondence to this author at: Centre de recherche du CHUM, Pavillon Saint-Luc, 264 René Lévesque Blvd East, Montréal, Quebec, H2X 1P1 Canada. Fax 514-281-2492.

	Abstract

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We have previously shown that the Nichols assay for intact parathyroid hormone (I-PTH) reacts with a non-(1–84) molecular form of PTH. This form behaves as a carboxy-terminal fragment and accumulates in renal failure, accounting for 40–60% of the measured immunoreactivity. We wanted to see whether this was a common event with other commercial two-site I-PTH assays. We thus compared the ability of three commercial kits [Nichols (NL), Incstar (IT), and Diagnostic System Laboratories (DSL)] to measure I-PTH in 112 renal failure patients and to detect hPTH(1–84) and non-(1–84)PTH on HPLC profiles of serum pools from uremic patients with I-PTH concentrations of 10–100 pmol/L. The behavior of synthetic hPTH(7–84), a fragment possibly related to non-(1–84)PTH was also compared with hPTH(1–84) in the three assays. The I-PTH concentrations measured with the three assays in the 112 uremic samples were highly related (r² 0.89, P <0.0001), and the values measured with NL were, on average, 23% higher than IT. Values measured with DSL were 23% and 56% higher than IT for values less than and more than 40 pmol/L, respectively. The three assays detected two HPLC peaks on four different profiles corresponding to hPTH(1–84) and non-(1–84)PTH. This last peak represented 36 ± 8.4% of the immunoreactivity with NL, 24 ± 5.5% with IT, and 25 ± 2.8% with DSL (NL vs IT or DSL: P <0.05). These differences were confirmed by a 50% lower immunoreactivity to hPTH(7–84) compared with hPTH(1–84) for IT and DSL but not for NL. These results suggest that most of the two-site I-PTH assays would cross-react with non-(1–84)PTH material, thus explaining about one-half of the 2–2.5 x higher I-PTH concentrations reported in uremic patients without bone involvement than in subjects without uremia.

	Introduction

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The development of immunoradiometric two-site assays for human intact parathyroid hormone (I-PTH) has greatly simplified PTH measurements in renal failure (1)(2)(3)(4). This simplification has been attributed to the improved specificity of these assays; they react only with the intact, biologically active form of the hormone (5)(6) and not with the inactive hormone fragments that are known to accumulate in renal failure (7)(8). However, in uremic patients these highly specific assays have measured a 2.5-fold increase in the nonsuppressible fraction of I-PTH compared with healthy subjects (9)(10)(11)(12)(13)(14). Moreover, I-PTH concentrations measured in uremic serum apparently overestimated PTH-related bone abnormalities also by a factor of 2–2.5 (15). The presence of circulating inhibitors of PTH has been proposed as one of the potential causes for these differences (16)(17).

We have already shown that when serum from healthy individuals and uremic patients was fractionated by HPLC, two immunoreactive peaks could be detected by the Nichols two-site I-PTH assay (18)(19). One peak was shown to comigrate with synthetic hPTH(1–84), and a second, more hydrophilic peak, was shown to accumulate in renal failure and accounted for 40–60% of the total immunoreactivity in these patients compared with 10–20% in healthy individuals (18).

The principal objectives of this study were to determine if this phenomenon could be observed with different I-PTH assays, and furthermore, if this could explain some of the reported discrepancies (2)(16)(17) that are regularly observed when different I-PTH assays are compared in uremic patients.

	Materials and Methods

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subjects
Specimens (112) from renal failure patients were used for this study. All samples were kept at -70 °C and were thawed only once for PTH measurement with three different I-PTH assays on the same day and by the same technician.

assays
Three commercial two-site I-PTH assays were tested: Allegro Intact PTH (Nichols Institute), N-Tact PTH SP (Incstar), and Active Intact PTH [Diagnostic System Laboratories (DSL)]. These three assays are almost identical with respect to the type of capture and signal antibodies used (affinity-purified goat polyclonal, anti-carboxyterminal capture antibody and anti-aminoterminal signal antibody), the tracer (I), incubation conditions (22 h at room temperature), the calibration material (synthetic hPTH(1–84) in a serum base), accuracy (both intraassay and interassay), reference values, and so forth. The major differences were that the DSL assay uses antibody-coated tubes instead of plastic beads and that this same assay has a lower sensitivity claim (0.6 pmol/L vs 0.1 and 0.07 pmol/L for the Nichols and Incstar assays, respectively). Each assay was run according to the manufacturer's protocol, using their own calibration material. About two-thirds of the samples were run in duplicate; one-third were run as singletons because of limited volumes of serum. The reactivity of each assay was also tested with pools of nondiseased human serum supplemented with synthetic hPTH(1–84) and hPTH(7–84) (Bachem).

chromatographic separations
Pools of serum from up to five patients with similar I-PTH concentrations or serum from single individuals were extracted on Sep-Pak Plus C₁₈ cartridges (Waters Chromatographic Division), and then chromatographed on a C₁₈ µ-Bondapak analytical column (Waters) using a discontinuous acetonitrile gradient (150–450 mL/L acetonitrile in 1.0 g/L trifluoroacetic acid) as previously described (18)(19)(20). After evaporation and freeze-drying, each 1.5-mL fraction was reconstituted either in 7.0 g/L bovine serum albumin or in a nondiseased human serum pool. The I-PTH content of each fraction was then measured using the three assays calibrated with their respective calibration materials.

statistical analysis
HPLC profiles were analyzed by planimetry using Origin 3.5 peak-fitting software (Microcal Software). Standard regression analysis was done using GRAPHPAD Instat software (Graphpad Software). The differences between I-PTH concentrations in uremic sera in the three assays were analyzed by the Friedman nonparametric repeated measures test, followed by Dunn's multiple comparisons test. The differences of the areas under the HPLC curves were analyzed by ANOVA for repeated measurements (Graphpad).

	Results

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The I-PTH concentrations obtained in each of the three assays in 112 uremic samples are compared in Fig. 1 . Results were highly correlated (r 0.89) although absolute values differed from assay to assay. Overall, as outlined by slope values, DSL tended to give higher values and Incstar lower values than the Nichols assay. Differences were not uniformly distributed along the measuring range. When the results were separated into low (<20 pmol/L), medium, and high (>40 pmol/L) ranges (Table 1 ), the values in the low and high ranges in the Nichols assay averaged 29% higher than the values in the Incstar assay (24% overall); whereas the values in the low and medium ranges in the DSL assay were 24% higher than the values in the Incstar assay and 56% higher in the high range (45% overall).

View larger version (28K): [in this window] [in a new window]   Figure 1. Regression parameters for I-PTH measured in 112 uremic samples by three commercial assays: A, intercept; B, slope.

Fig. 2 shows the typical HPLC profiles observed at two different I-PTH concentrations (~60 pmol/L in the top chromatogram and ~100 pmol/L in the bottom chromatogram) in the three assays. The three assays reacted with two peaks of immunologically reactive PTH. A major, more hydrophobic peak comigrated with synthetic hPTH(1–84) (right arrow), whereas a minor, slightly less hydrophobic peak migrated before hPTH(1–84) and just ahead of hPTH(7–84) (left arrow). As shown on Table 2 , the percentage of the area under the curve for the non-(1–84) peak was ~45% higher (P <0.05) in the Nichols assay, and the area corresponding to hPTH(1–84) was 15% smaller than in the two other assays.

View larger version (20K): [in this window] [in a new window]   Figure 2. HPLC profiles of circulating I-PTH in two pools of uremic samples (top, ~60 pmol/L; bottom, ~100 pmol/L). Assays: Nichols (—), Incstar (... .), and DSL (- - -). Left arrow, hPTH(7–84) standard; right arrow, hPTH(1–84) standard.

To gain a better understanding of these differences, we next analyzed the immunoreactivity of hPTH(1–84) and hPTH(7–84), a commercially available molecule potentially structurally related to the non-(1–84)PTH peak. As shown on Fig. 3 , hPTH(1–84) and hPTH(7–84) reacted almost equimolarly in the Nichols assay, whereas hPTH(7–84) was only one-half as potent as hPTH(1–84) in the two other assays.

View larger version (25K): [in this window] [in a new window]   Figure 3. Immunoreactivity of hPTH(1–84) (solid lines) and hPTH(7–84) (broken lines) in the Nichols, Incstar, and DSL assays.

	Discussion

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We have already published data supporting the presence of non-(1–84)PTH material reacting with the Nichols I-PTH assay in healthy individuals (18) and in uremic patients (19). This has been confirmed by others (16)(17). The impact of this finding on the differences observed in I-PTH values by different assays in renal failure patients forms the basis of this study.

Our results first indicated (Fig. 1 and Table 1 ) that there were significant differences between the Nichols, DSL, and Incstar assays when uremic sera were measured. The assays were carried out under strict conditions: All measurements were taken for the same set of samples, on the same day, by the same technician, using the same freshly thawed samples previously kept under recognized stable conditions (-70 °C, single freeze-thaw cycle). Results were recalculated using different algorithms (spline, log-log) to guarantee that the calculations did not introduce a bias in the reported PTH concentrations (results not shown). Two separate comparisons were done, using kits from different lot numbers, with no variation in the observed results. Overall, the Incstar assay gave lower values than either the Nichols or DSL assay over the entire measuring range. Differences between the Incstar and Nichols assays were consistent with results observed after fractionation of uremic serum samples by HPLC and reactivity with the synthetic hPTH(7–84) fragment. The Incstar assay reacted 50% less with this hPTH(7–84) fragment than did the Nichols assay (Fig. 3 ). Because non-(1–84)PTH material constitutes 40–60% of the total immunoreactive I-PTH in uremic samples (19) and assuming that the material found in the non-(1–84) peak reacted similarly to hPTH(7–84), we would have expected a 20–25% difference between the Incstar and Nichols assays. This is the case over the entire measuring range (24%). For reasons that are not clear at this time, the DSL assay, with a reactivity towards hPTH(7–84) comparable with the Incstar assay, nevertheless gave results higher than the Incstar and even higher than the Nichols assay for values >40 pmol/L (Table 1 ). Differences in calibration procedures and matrix effects may have been a contributory factor. Reactivity to molecular forms of non-(1–84)PTH other than hPTH(7–84) also may have played a role.

Although they were purified by affinity chromatography, the polyclonal antibodies used as signal and capture antibodies in the three assays theoretically have the ability to react with hPTH fragments lacking a small number of amino acids at either end of the PTH molecule. Most amino-terminal-revealing antibodies presently available react against one or more epitopes located in the 14–34 region of the PTH molecule (21). Amputation of a few amino acids at the amino-terminal end of PTH, relatively remote from the 14–34 region should, therefore, not prevent immunoreactivity in these assays. According to the respective inserts, the cross-reactivity of the three kits evaluated in this work were checked using PTH fragments lacking a very large number of amino acids at the N-terminus: hPTH-(39–84), hPTH-(53–84), hPTH-(39–68), and hPTH-(44–68) (5)(6). None of the kits apparently was checked with fragments missing the very first N-terminal amino acids, such as the hPTH(7–84) fragment tested in this study, perhaps because of limited commercial availability at the time of market launch.

The clinical consequences of the cross-reactivity of I-PTH assays with non-(1–84)PTH are twofold. First, the molecular entities migrating in the non-(1–84)PTH peak are probably devoid of at least some of the very first N-terminal amino acids (17) necessary for adenylate cyclase activation (22). Therefore, the three PTH assays measured inactive fragments and overestimated PTH secretion by 80–120%, depending on the assay. This probably contributes to the 2- to 2.5-fold higher "normal values" observed in uremic patients in the absence of PTH-mediated bone involvement (15). Second, hPTH material migrating in the non-(1–84) peak has possibly retained the ability to bind to PTH receptors. The binding portion of PTH and the major epitope for preparing antibodies against the PTH amino-terminal end are located in the same region of the PTH molecule (21)(22). By binding with the receptors, non-(1–84)PTH could, therefore, act as a physiological antagonist of PTH and thus contribute to the apparent PTH resistance (and accompanying increased PTH secretion) observed in uremic patients (7)(8)(9)(10)(11)(12). Further studies will be required to elucidate this last point and also to evaluate the effect of differences in the calibration material, now that we have shown that large non-(1–84) PTH fragments are measured differently by various I-PTH assays. Because there is no prima facie reason to think that antibodies to the endmost N-terminal portion of PTH cannot be generated, the development of a "truly" I-PTH assay remains a desirable goal.

	Acknowledgments

 
We thank Manon Livernois for assistance with the manuscript. This work was made possible by grant MA-7643 from the Medical Research Council of Canada.

	References

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