Measuring Parathyroid Hormone (PTH) in Patients with Oxidative Stress – Do We Need a Fourth Generation Parathyroid Hormone Assay?

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.

LC-MS candidate reference methods for the harmonisation of parathyroid hormone (PTH) measurement: a review of recent developments and future considerations

“…Despite the improved performance of these assays in some patient groups [35], some anomalous results were still reported [36][37][38].…”

“…Amongst other possible degradation products such as deamidation of asparagine (ASN, N) residues, methionine (MET, M) oxidation is likely to be the most prevalent. In vitro studies using high-resolution MS to identify oxidation products, following exposure of these compounds to peroxides, have demonstrated that the methionine residues at positions 8 and 18 of PTH1-34 and PTH1-84 can become oxidised [38,54]. Methionine 8 oxidises more slowly than methionine 18 [55], and both methionine residues can be singly or doubly oxidised (to sulphoxides and sulphones, respectively).…”

“…Methionine 8 oxidises more slowly than methionine 18 [55], and both methionine residues can be singly or doubly oxidised (to sulphoxides and sulphones, respectively). It is also well established that all oxidised PTH variants are less biologically active than non-oxidised PTH [38,[56][57][58][59][60]. With these findings considered in the context of clinical PTH measurement, Hocher et al [38,61] raised a monoclonal antibody against oxidised PTH, and immobilised this antibody in microcolumns.…”

“…It is also well established that all oxidised PTH variants are less biologically active than non-oxidised PTH [38,[56][57][58][59][60]. With these findings considered in the context of clinical PTH measurement, Hocher et al [38,61] raised a monoclonal antibody against oxidised PTH, and immobilised this antibody in microcolumns. High-resolution MS was used to demonstrate the efficiency of the antibody prior to use.…”

“…These affinity columns were then used to remove oxidised PTH in clinical samples from haemodialysis patients prior to analysis of non-oxidised PTH species using immunoassay (second-generation, Roche PTH assay). It was demonstrated that a large, yet highly variable, proportion ( > 90% in some cases) of the total PTH in these samples was present in an oxidised form [38,61].…”