Using Mass Spectrometry to Improve PTH Identification and Test Accuracy

Parathyroid hormone (PTH) is associated with a range of kidney- and bone-related diseases. Its levels within the blood are therefore tested to monitor for these diseases in patients. However, traditional assay techniques have been shown to not account for all the PTH variants that are known to occur within the blood. Mass spectrometry techniques are now being developed to produce more accurate and responsive assays of PTH.

PTH is essential in regulating calcium levels within the body. It is produced and secreted into the blood from the parathyroid glands and is taken up in a variety of tissues. Very high levels are taken up in kidney tubules and bone cells because calcium is very important in the functioning of these tissues.¹ Abnormal PTH in the blood (hyper- or hypoparathyroidism) can therefore cause problems with the kidneys and bones, and PTH assays are done to help diagnose hypo-/hyperparathyroidism, as well as monitor chronic kidney disease.²

The amount of PTH is not the only factor associated with pathological conditions. PTH also has various fragmented forms, and although these occur in normal individuals¹, end-stage renal-disease patients often have large quantities of PTH fragments in comparison to intact PTH (PTH1-84) (Figure 1).³

Traditionally, PTH assays have used specific antibodies that bind to PTH (known as first-generation assays) in order to identify the quantity of PTH within a patient’s blood.¹ However, these assays are unable to identify the various forms of PTH because of the difficulty in producing sufficiently specific antibodies.¹ Therefore, second-generation assays were developed that use two different antibodies that bind to the start and end of the protein hormone in an attempt to identify the quantity of only intact PTH. However, these assays were found to also measure PTH forms with an amino-acid deletion at the start of the protein, mainly PTH7-84.^4,5 This, again, was related to the specificity of the antibodies used.

Therefore, new third-generation assays are beginning to be developed that use mass spectrometry in order to measure the levels of various forms of circulatory PTH fragments.¹ Work by Lopez et al.⁶ used a LTQ-Orbitrap XL Mass Spectrometer (Thermo Scientific) and identified five previously unknown clinical variants of PTH using samples from healthy individuals and renal-failure patients. From this work quantitative selected reaction monitoring (SRM) assays were developed on a TSQ Vantage Triple-Quadrupole Mass Spectrometer (Thermo Scientific) for a variety of PTH forms. Using top-down protein analysis, the full, intact PTH was identified, as well as a form commonly mistaken for intact PTH in second generation assays, PTH7-84, and a much shorter fragmented form, aa 34-84.

These newly identified PTH forms may be of useful clinical value in the future, or they could be “analytical nuisances” with the potential to confound PTH assays⁶. Either way, the development of accurate representations of PTH quantities in the blood are vitally important for reliable PTH assays in the future.

References

1. Vieira, J.G., (2012) ‘PTH assays: Understanding what we have and forecasting what we will have‘. Journal of Osteoporosis, Published online April 3, 2012. doi: 10.1155/2012/523246

2. Aloia, J.F., Feuerman, M., and Yeh, J.K. (2006) ‘Reference range for serum parathyroid hormone‘, Endocrine Practice, 12 (2), (pp. 137-144)

3. Vieira, J.G., Kunii, I.S., Ohe, M.N., and Carvalho, A.B. (2009) ‘Heterogeneity of carboxyl-terminal parathyroid hormone circulating forms in patients with hyperparathyroidsism due to end stage renal disease.’ Arquivos Brasileiros de Endocrinologia e Metabologia, 53 (9), (pp. 1074-1078)

4. Brossard, J.H., Cloutier, M., Roy, L., Lepage, R., Gascon-Barré, M., and D’Armour, P. (1996) ‘Accumulation of a non-(1-84) molecular form of parathyroid hormone (PTH) detected by intact PTH assay in renal failure: importance in the interpretation of PTH values‘. Journal of Clinical Endocrinology and Metabolism, 81 (11), (pp. 3923-3929)

5. Lepage, R., Roy, L., Brossard, J.H. et al. 1998. ‘A non-(1-84) circulating parathyroid hormone (PTH) fragment interferes significantly with intact PTH commercial assay measurements in uremic samples‘. Clinical Chemistry, 44 (4), (pp. 805-809)

6. Lopez, M.F., Rezai, T., Sarracino, D.A., Prakash, A., Krastins, B., Athanas, M., Singh, R.J., Barnidge, D.R., Oran, P., Borges, C., and Nelson, R.W. (2010) ‘Selected reaction monitoring – mass spectrometric immunoassay responsive to parathyroid hormone related variants‘. Clinical Chemistry, 56 (2), (pp. 281-290)