Chemical Characterization and Quantification of Circulating Intact PTH and PTH Fragments by High-Resolution Mass Spectrometry in Chronic Renal Failure

Abstract

Background The precise concentrations of full-length parathyroid hormone (PTH1-84) and the identity and concentrations of PTH fragments in patients with various stages of chronic renal failure are unknown. Methods We developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method to characterize and quantify PTH1-84 and PTH fragments in serum of 221 patients with progressive renal dysfunction. Following capture by matrix-bound amino-terminal or carboxyl-terminal region-specific antibodies and elution from matrix, PTH1-84 and PTH fragments were identified and quantitated using LC-HRMS. PTH was simultaneously measured using an intact PTH (iPTH) immunoassay. Results Full-length PTH1-84 and 8 PTH fragments (PTH28-84, 34-77, 34-84, 37-77, 37-84, 38-77, 38-84, and 45-84) were unequivocally identified and were shown to increase significantly when an eGFR declined to ≤17-23 mL/min/1.73m2. Serum concentrations of PTH1-84 were similar when measured by LC-HRMS following capture by amino-terminal or carboxyl-terminal immunocapture methods. In patients with an eGFR of <30 mL/min/1.73 m2, serum PTH concentrations measured using LC-HRMS were significantly lower than PTH measured using an iPTH immunoassay. PTH7-84 and oxidized forms of PTH1-84 were below the limit of detection (30 and 50 pg/mL, respectively). Conclusions LC-HRMS identifies circulating PTH1-84, carboxyl-terminal PTH fragments, and mid-region PTH fragments, in patients with progressive renal failure. Serum PTH1-84 and its fragments markedly rise when an eGFR decreases to ≤17-23 mL/min/1.73 m2. PTH concentrations measured using LC-HRMS tend to be lower than those measured using an iPTH immunoassay, particularly in severe chronic renal failure. Our data do not support the existence of circulating PTH7-84 and oxidized PTH1-84.

Proteoforms and their expanding role in laboratory medicine

“…Clinically, PTH is measured via two-site immunoassays; however, in vivo proteolytic cleavage products of PTH can interfere with these assays. Many in vivo fragments of PTH have been identified, including N-terminally truncated proteoforms spanning residues 7–84, 28–84, 34–84, and 38–84, as well as both C and N-terminally truncated proteoforms: 34–77, 38–77, and 45–34 [ [87] , [88] , [89] , [90] ]. Questions pertaining to such fragments include whether they circulate in high abundance, whether they are active or inactive fragments, and their relation to physiology and clearance (e.g., PTH 1–84 has a half-life of ∼5 min, whereas some fragments have half-lives in the range of 24–36 h) [ 87 , [91] , [92] , [93] ].…”

“…In a recent study examining the primary structure and concentration of PTH fragments, an intact proteomic discovery analysis was used to sequence the PTH fragments circulating in patients with renal failure [ 90 ]. This method used immunoenrichment with two anti-PTH antibodies, one against the C-terminus (epitope within residues 44–84) and one against the N-terminus (immunogen/epitope not specified).…”

“…Further, when quantifying PTH in individuals with severe renal failure, where increased PTH proteolytic fragmentation is expected, a method comparison between the MRM assay and a third-generation immunoassay demonstrated that the MRM assay had 58% lower PTH 1–84 concentration relative to the immunoassay. While the third-generation immunoassay did not demonstrate significant cross-reactivity with N-terminally cleaved PTH 28–84, 34–84, 37–84, 45–84, or 48–84, it did have nearly 100% cross-reactivity with synthetic PTH 7–84 [ 90 ].…”

Novel Insights into Mechanisms of Intestinal Phosphate Absorption in Patients with Chronic Kidney Disease

“…Hyperphosphatemia is generally not evident until the eGFR falls below 30 ml/min per 1.73 m, 1 due to a compensatory reduction in renal tubular phosphate reabsorption mediated by elevated fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH) levels in CKD. 1,2 Moreover, renal production of 1,25-dihydroxyvitamin D [1,25(OH) 2 D] is diminished owing to increased FGF23 levels, decreased nephron numbers, and an increase in serum phosphate concentrations due to phosphate retention. Because 1,25(OH) 2 D enhances intestinal phosphate transport, reduced intestinal phosphate absorption is expected as kidney function and 1,25(OH) 2 D concentrations decline.…”

“…1 In addition, patients rank PTDM as a leading cause of concern. 2 Therefore, exploring strategies that mitigate the risk of developing diabetes after transplantation is strongly advocated by both international consensus statements 3 and national guidelines. 4 However, interventional studies targeting prevention of PTDM as the primary study outcome are limited.…”