Parathyroid hormone is involved in calcium homeostasis through interactions with vitamin D. Because intact and truncated forms of parathyroid hormone (PTH) vary in their biological activity, assays that can accurately quantify the ratio of intact hormone to its fragments are of increasing significance in the diagnosis of endocrine, renal and bone diseases. Vitamin D and its metabolites circulate tightly bound to vitamin D-binding protein (DBP). Because DBP concentrations are altered in pregnancy, liver and renal diseases and also show genetic variations in different ethnic groups, total vitamin D in serum can be misleading. In addition, both calcium and vitamin D metabolites can decrease the secretion of PTH.
Previously, we developed multiplexed mass spectrometric immunoassay (MSIA)SRM assays for PTH that allow quantification of four fully-tryptic monitoring peptides (that span the entire PTH sequence) and two semi-tryptic variant specific peptides1. Using this approach, it is possible to monitor intact PTH and also the degree of N-terminal fragmentation.
In this study, the objective was to develop a multiplexed, MSIA-SRM-based targeted assay for PTH and DBP. We applied this MSIA-SRM assay and a commercially available immunoassay to a cohort of 500 clinical samples from a variety of different patient groups including renal disease, cancer, vitamin D deficiency and other conditions that can affect calcium homeostasis. The results demonstrated excellent assay linearity (R2=0.900.99) with sensitivity for analytes in the published clinical ranges and limits of detection in the pg/ml range. Comparison of the PTH MSIA SRM assay with the commercial ELSA assay demonstrated good correlation in normal subjects but important differences in renal failure. There were also some unusual fragments seen in clinical samples, not previously reported in the literature.
1. Lopez MF, Rezai T, Sarracino DA, Prakash A, Krastins B, Athanas M, Singh RJ, Barnidge DR, Oran P, Borges C & Nelson RW. Selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants. Clin Chem 2010 56 28190.
Declaration of funding: Funding of reagents and consumables were from Thermo Scientific US.