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. 2012;7(7):e40242.
doi: 10.1371/journal.pone.0040242. Epub 2012 Jul 6.

Measuring parathyroid hormone (PTH) in patients with oxidative stress--do we need a fourth generation parathyroid hormone assay?

Berthold Hocher  1 , Franz Paul Armbruster, Stanka Stoeva, Christoph Reichetzeder, Hans Jürgen Grön, Ina Lieker, Dmytro Khadzhynov, Torsten Slowinski, Heinz Jürgen Roth
Affiliations

Measuring parathyroid hormone (PTH) in patients with oxidative stress--do we need a fourth generation parathyroid hormone assay?

Berthold Hocher et al. PLoS One. 2012.

Abstract

Oxidation of PTH at methionine residues results in loss of biological activity. PTH may be oxidized in patients with renal disease. The aim of this study was to develop an assay considering oxidation of PTH. Oxidized hPTH was analyzed by high resolution nano-liquid chromatography coupled to ESI-FTT tandem mass spectrometry (nanoLC-ESI-FT-MS/MS) directly and after proteolytic cleavage. The oxidized hPTH(1-84) sample shows TIC-peaks at 18-20 min and several mass peaks due to mass shifts caused by oxidations. No significant signal for oxidized hPTH(1-84) species after removal of oxidized PTH molecules by a specific column with monoclonal antibodies (MAB) raised against the oxidized hPTH was detectable. By using this column in samples from 18 patients on dialysis we could demonstrate that measured PTH concentrations were substantially lower when considering oxidized forms of PTH. The relationship between PTH concentrations determined directly and those concentrations measured after removal of the oxidized PTH forms varies substantially. In some patients only 7% of traditionally measured PTH was free of oxidation, whereas in other patients 34% of the traditionally measured PTH was real intact PTH. In conclusion, a huge but not constant proportion of PTH molecules are oxidized in patients requiring dialysis. Since oxidized PTH is biologically inactive, the currently used methods to detect PTH in daily clinical practice may not adequately reflect PTH-related bone and cardiovascular abnormalities in patients on dialysis.

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Conflict of interest statement

Competing Interests: Dr. Hocher, Dr. Armbruster and Dr. Roth submitted a patent application. The title is “MEANS AND METHODS OF MEASURING PARATHYROID HORMONE IN PATIENTS SUFFERING FROM OXIDATIVE STRESS.” The application number is 12156441.3. This application does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. Dr. Armbruster is the CEO of Immundiagnostik AG, Bensheim, Germany. Dr. Stoeva and Dr. Grön are research employees of Immundiagnostik AG, Bensheim, Germany. Dr. Roth is a research employee of the Department of Endocrinology/Oncology, Limbach Laboratory, Heidelberg, Germany. This does not alter the authors’ adherence to all the PLoS ONE polices on sharing data and materials.

Figures

Figure 1
Figure 1
A: Under conditions of oxidative stress the methionine residues at position 8 and 18 may be oxidized to methionine sulfoxide and methionine sulfone. Oxidation to methionine sulfoxide is reversible, whereas the second oxidation step to methionine sulfone is irreversible. Oxidized PTH changes its 3-dimesional structure. This blocks the interaction of PTH with its receptor. B: Schematic diagram of the full length PTH(1–84) molecule (“bioactive” intact PTH). Oxidation at position Met 8 and/or Met 18 (red) alters the receptor binding site of PTH. Oxidized PTH does not bind the PTH receptor anymore and is thus biologically inactive. (see: E. Blind, Clin. Lab. 2008;54∶439-446, reference 3).
Figure 2
Figure 2. Non-digested oxidized synthetic hPTH(1–84)ox.
A: NanoLC-ESI-FTMS total ion chromatogram. B: Magnified summed FTMS spectrum for retention time interval 18.30–20.50 minutes. Several different charged analyte ions were detected.
Figure 3
Figure 3. Flow through fraction of non-digested oxidized synthetic hPTH(1–84)ox from the affinity column.
A: NanoLC-ESI-FTMS total ion chromatogram. B: Magnified summed FTMS spectrum for retention time interval of 16.50–18.50 minutes. The spectrum does not show any analyte masses which belong to PTH or oxidized PTH.
Figure 4
Figure 4. Eluate from the affinity column of non-digested oxidized synthetic hPTH(1–84)ox.
A: NanoLC-ESI-FTMS total ion chromatogram. B: Magnified summed FTMS spectrum for retention time interval of 16.50–18.50 minutes. Several different charged analyte ions of PTH eluate were detected.
Figure 5
Figure 5. Comparison of the enlarged spectra of the starting material, non-digested oxidized synthetic hPTH(1–84)ox ( Fig.1B ), and the eluate from the affinity column of non-digested oxidized synthetic hPTH(1–84)ox (Fig. 3B).
Figure 6
Figure 6. Basic principles of the new assay system for detection of intact and real intact PTH in human samples.
The new detection process for real intact bioactive PTH consists of 2 steps: Firstly, any oxidized forms of oxidized PTH at position Met8 and/or position Met18 will be removed from the probe by a specific affinity chromatography column. The column contains monoclonal rat/mouse antibody (MAB) raised against the hPTH(1–34)oxidized fragments. These antibodies are able to remove any oxidized forms of PTH. In the second step the remaining non-oxidized PTH will be analyzed in conventional 2-site “sandwich” immunoassay systems. The antibody on the left (capture antibody) is bound to solid phase. The antibody on the right (label antibody) relays the signal. These antibodies must bind different sites on the PTH analyte to produce a positive result in the assay.
Figure 7
Figure 7. We measured intact PTH using the intact PTH assay as described in the method section in 18 patients on dialysis (blue bars), for further detail see also table 2 .
When removing the oxidized forms of PTH from the sample as illustrated in figure 6, the results were completely different (red bars). The effect of oxidation of PTH is highly variable among these patients requiring dialysis. There is only a very weak correlation between traditionally measured PTH and PTH data considering the oxidation of this hormone.
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