ECTS2014 Poster Presentations Bone development/growth and fracture repair (55 abstracts)
1Kings College Hospital, London, UK; 2ThermoFisher Scientific BRIMS Center, Cambridge, Massachusetts, USA.
The analysis of intact parathyroid hormone (PTH) (PTH184) is useful in the diagnosis of hyper- and hypocalcaemia, hyperparathyroidism, and in the prevention of bone mineral disorders in renal patients. However, the analysis is complicated by the presence of PTH fragments. These are especially prone to accumulation in renal failure, and cross-react differently with different immunoassays, including the most recent, third-generation immunoassays. As such, large variability exists between different commercially available assays, as well as reference ranges for guiding treatment. This variability may be considered a critical governance issue in renal patients, and harmonisation of testing is urgently required.
Following the 50th anniversary of the introduction of the first PTH R/A last year, there has been little standardisation of PTH testing, despite our increased understanding of PTH biology. Recently, mass spectrometric methods have been developed as reference methods for harmonising the quantitation of PTH184, but even these methods are subject to significant interferences from variants including oxidised PTH. In this work, we report the use of mass spectrometric immunoassay for the identification of phosphorylated PTH in clinical samples. The phosphorylated tryptic peptide, PTH1420 (HLNS(P)MER), was detected in 58 (16%), and quantified in 15 (4%) of samples (LOD and LLoQ 10 and 30 pg/ml respectively). The median (range) concentration in samples with quantifiable phosphorylated PTH1420 was 52 (32767) pg/ml, compared to a median (range) concentration of 287 (431883) pg/ml for the equivalent non-phosphorylated PTH1420 peptide (LLoQ 30 pg/ml). There was no correlation between concentrations of HLNSMER and HLNS(P)MER (R2=0.01).
Further clinical investigation into the biological activity of these variants is necessary, plus further analytical work including i) the use of high-resolution mass spectrometry, and ii) the analysis of PTH without prior protease digestion, is required before mass spectrometry-based approaches can be considered as reference methods against which other methods should be harmonised.