Parathyroid hormone (PTH) plays a critical role in the regulation of circulating blood calcium levels, and serves as a biomarker of secondary hyperparathyroidism associated with the diagnosis of disorders such as vitamin D deficiency or chronic kidney disease (CKD), and for monitoring the effectiveness of treatment e.g., hemodialysis. Immunochemiluminescent assay of full length (i.e., 1-84) PTH has historically been problematic due to variable standardization, cross reactivity with truncated PTH fragments (e.g., 7-84), and variable correlations with biological parameters.  A recent report that PTH may be oxidized in vivo further complicates these issues, since oxidized PTH is biologically inactive.

To address these challenges, an immunocapture liquid chromatography–tandem mass spectrometry (‘immuno-LC-MS/MS’) analysis strategy was developed and optimized, together with a novel dual isotope-labeled internal standard approach (i.e., ¹⁵N-labelled full length and truncated PTH and ¹³C₆¹⁵N isotope-labelled oxidized PTH tryptic peptides), in order to (i) quantitatively determine the nature of circulating full length and truncated PTH and their individual oxidized isoforms, (ii) differentiate between in vivo and ex vivo oxidative modifications, and (iii) examine the effects of PTH oxidation on current immunochemiluminescent PTH assays. First, using a series of purified full length, truncated and individual oxidized isoforms of PTH spiked into depleted plasma, the recovery of the overall analysis strategy was determined to be 66%, with a wide linear range for detection from 5-2000 pg/mL.

Then, two series of plasma samples from patients on hemodialysis were analyzed.  In Set A, oxidized full length and truncated PTH were commonly observed, with oxidation ranging from 0 to 80% of the total PTH observed, and with 34 of the 41 samples containing more than 10% oxidization.  In general, there was more oxidized PTH 7-84 than oxidized PTH 1-84.  In contrast, in Set B, only minimal amounts of oxidized PTH peptides were observed, with only 3 of 29 samples containing >10% oxidation. The marked difference in oxidized PTH peptides between Set A and Set B suggests that oxidation of PTH may be occurring ex-vivo.  Further studies are required to fully define the possibility and origin of ex-vivo oxidation.  The immuno LC-MS/MS results correlated extremely well with the assay of PTH 1-84 and less well with other “intact PTH” assays.  The effects of oxidation on the results obtained using current intact PTH assays may represent an additional factor contributing to the variability of PTH assay results and their correlation with biological parameters.