Abstract :
[en] Introduction: Chronic kidney disease (CKD) affects approximately 10% of the global population and is characterized by a progressive and irreversible decline in glomerular filtration rate (GFR). According to KDIGO guidelines, monitoring of these patients relies on the assessment of various biomarkers, including parathyroid hormone (PTH). PTH is a peptide hormone composed of 84 amino acids, secreted by the parathyroid glands in response to a decrease in extracellular calcium concentration. In patients with CKD, mineral disorders often lead to hypocalcemia, which triggers an increased secretion of PTH. However, some of these patients exhibit abnormally high PTH levels, suggesting resistance to the hormone’s action, resulting in bone demineralization and an increased risk of fractures. The mechanisms underlying PTH resistance in CKD patients, as well as its interindividual variability, remain poorly understood. One hypothesis suggests that this resistance may be related to the presence of PTH fragments, eight of which have been identified in human serum by Kritmetapak et al.
Objective: This work aims to develop a method for quantifying parathyroid hormone fragments using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS).
Method: We developed an LC-MS/MS method to quantify four of the eight PTH fragments (PTH 38–77, PTH 38–84, PTH 37–84, and PTH 34–84). Serum samples were prepared by solid-phase extraction (SPE MAX) before being analyzed by UPLC-MS/MS using Shimadzu NEXERA X2® HPLC H-Class Plus chromatographic system coupled with a AB SCIEX® QT-6500 mass spectrometer.
Results: Chromatographically, four PTH peptides were successfully separated, ensuring sufficient selectivity. We performed a proof-of-concept study by quantifying these four fragments in pooled serum samples from patients categorized according to their eGFR (<15, 15–30, 30–45, and 45–60 mL/min/1.73 m²). We observed the same trends as those reported by Kritmetapak, namely an increase in the serum concentrations of C-terminal fragments as eGFR decreases. We also analyzed serum samples from patients with the same eGFR and found that fragment concentrations varied between individuals up to 13 times. This suggests that the concentrations of these fragments do not depend solely on eGFR and may reflect varying degrees of PTH resistance.
Conclusion: A quantification method by LC-MS/MS has been developed for four of the eight PTH fragments. We aim to further optimize and validate this method by including the remaining four fragments as well as the intact PTH, in order to obtain a comprehensive overview of PTH metabolism in a single acquisition. Regarding future perspectives, if these fragments are indeed involved in the mechanism of PTH-induced bone resistance, their accumulation suggests that they could serve as valuable biomarkers for monitoring these patients. Our LC-MS/MS method will allow their quantification, after which they could be eliminated, for example, by optimizing the dialysis modality or the type of membrane used. Thus, the LC-MS/MS quantification method would provide clinicians with the ability to personalize patient management, improve the prevention of fractures—which are often fatal—and reduce the substantial associated costs.
