The effect of different protease inhibitors on stability of parathyroid hormone, insulin, and prolactin levels under different lag times and storage conditions until analysis

Abstract

Introduction: Proteolytic cleavage through proteases affects peptide hormone levels, which is of particular significance when the time interval between sampling and analysis is prolonged. We evaluated the stability of parathyroid hormone, insulin, and prolactin molecules (i) with different protease inhibitors such as K₂ EDTA, aprotinin, and protease inhibitor cocktail (PIC), (ii) with different lag times (6-72 hours), and (iii) under different storage temperatures (4°C vs room temperature [RT]) until analysis.

Materials and methods: Blood samples were collected into 2 sets of 5 Vacutainer^® tubes (Becton Dickinson) from 10 healthy adults. Tubes 1 and 2 were plain gel separator tubes. Tubes 3, 4, and 5 contained PIC (1%), aprotinin (500 KIU/mL), and K₂ EDTA, respectively. After centrifugation at 1300 g for 10 minutes, PIC added to tube 2 of each set. Samples were analyzed and then one set was stored at 4°C, whereas the other at RT until analysis at 6, 24, 48, and 72 hours. Hormone levels were determined with electrochemiluminescence immunoassay (ModularE170; Roche Diagnostics). The results were compared with desirable bias limits (DBL) from Westgard QC database.

Results: Insulin at RT decreases exceeding the DBL starting from 24 hours and K₂ EDTA preserved insulin. PTH exceeded the DBL at RT for 48 hours or longer and PIC addition after centrifugation inhibited its degradation. Prolactin remained stable in all tested conditions. All parameters in the plain gel separator tubes remained within DBL when stored at 4°C until 72 hours.

Conclusions: Different proteases may degrade peptide hormones and measures should be taken to counteract these effects especially if there is a delay before analysis.

Keywords: insulin; parathyroid hormone; pre-analytical phase; prolactin; protease inhibitors.

Figure 1

Median insulin, parathyroid hormone and prolactin concentrations over time under various conditions expressed as percentage of the concentration at time (T)=0. RT, room temperature; Post PIC, protease inhibitor cocktail addition after centrifugation; Pre PIC, protease inhibitor cocktail addition before centrifugation