Degradation and Stabilization of Peptide Hormones in Human Blood Specimens.
Author(s): Yi J, Warunek D, Craft D
Publication: PLoS One, 2015, Vol. 10, Page e0134427
PubMed ID: 26222180 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to assess the stability of the active forms of select circulating peptide gut hormones (glucagon-like peptide 1, GLP-1; glucose-dependent insulionotropic polypeptide, GIP; oxyntomodulin, OXM), and glucagon ) in whole blood, serum, and plasma collected and stored pre- or post-centrifugation in tubes containing different anticoagulants with or without protease inhibitors.
Conclusion of Paper
Evidence of proteolytic activity was observed in serum, EDTA-, citrate- and heparin-plasma after a post-centrifugation delay at room temperature. Incubating EDTA plasma on wet ice as opposed to room temperature during the delay only partially attenuated the effects of the post-centrifugation delay on GIP-1 levels. However, all peptide gut hormones were stable for up to 72 h at room temperature in plasma from P800 tubes that contained a proprietary protease inhibitor cocktail. A pre-centrifugation delay also resulted in declines in GIP-1 among non-anticoagulated blood and EDTA plasma stored on wet ice or at room temperature. In response to a pre-centrifugation delay, GLP-1 levels also declined in plasma when blood was collected in P800 tubes, however the declines were more modest and calculated half-life was much longer than were observed for blood collected in EDTA tubes (>72 h vs. 20.6 h). The authors conclude that proteases remain active in blood-derived specimens both before and after centrifugation, and can only be attenuated by the inclusion of protease inhibitors in the blood collection tube.
Studies
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Study Purpose
The purpose of this paper was to assess the post-centrifugation stability of the active forms of select circulating peptide gut hormones in serum, and plasma collected using different anticoagulant with and without protease inhibitors . Blood was collected from at least six healthy volunteers into different tube types, centrifuged for 15 min for 2,500 x g, and then 10 µl from each patient was pooled in a new tube that was then spiked with the target peptide. The pooled, spiked specimen was then subjected to a room temperature delay of 0-96 h in a temperature controlled chamber or on ice. Specimens were analyzed immediately or frozen at -80°C. Peptide targets included glucagon-like peptide 1 (GLP-1), glucose-dependent insulionotropic polypeptide (GIP), oxyntomodulin (OXM), and glucagon.
Summary of Findings:
In EDTA plasma active forms of GLP-1 (G36A, G37) began to decline after a post-centrifugation delay of 4 h at room temperature, which corresponded to an increase in abundance of their respective fragments (G36A-2N, G37-2N). The increase in G36-2N and G37-2N, which are both lacking two N-terminal residues, suggests that DPP-IV remains active in centrifuged EDTA plasma. However, subsequent declines in G36-2N and G37-2N signal intensities in EDTA plasma after 72 h at room temperature, indicated other peptidases are also active. Similar GLP-1 stabilities were reported for citrate- and heparin-plasma, as well as GIP in serum specimens compared to that of EDTA plasma. Inclusion of a protease inhibitor cocktail optimized to target DD-IV and other peptidases in blood collection tubes (P700 and P800 tubes) served to stabilize GLP-1 active forms G36A and G37, delaying their decline and the appearance of their fragments (G36A-2N and G37-2N) until 72 h. The calculated half-life of G36A was extended from 6 h in EDTA plasma to >96 h when the protease inhibitor was included (B700 and B800 tubes). ELISA targeting G36A confirmed mass spectrometry findings, as its concentration remained stable for 72 h in plasma collected in B700 or B800 tubes compared to EDTA plasma that exhibited a decline within 5 h. The authors note the decline in G36A in EDTA plasma occurred rapidly, dropping to 50% of the concentration observed in B800 tubes within 20 min. This decline was partially attenuated when EDTA plasma was stored on ice during the post-centrifugation delay, reducing the magnitude of decline to 20% with a 20 min delay, and extending the projected half-life to 15 h. Comparable results were obtained regardless of whether P800 plasma was stored on wet ice or at room temperature during the post-centrifugation delay. Similar trends were observed for other peptides (oxyntomodulin, OXM; glucagon) cleaved from the same protein (proglucagon). The active form of OXM began to decline after a 24 h post-centrifugation delay, which coincided with the first appearance of its fragment in EDTA plasma, while levels of OXM in P800 plasma remained stable for 72 h. The calculated half-life of glucagon was longer in P800 compared to EDTA plasma tubes, increasing from 20.4 h to 45.6 h.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Peptide MALDI-TOF MS Peptide Radioimmunoassay Peptide Immunoassay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant EDTA
Citrate
Heparin
None
Biospecimen Acquisition Type of collection container/solution EDTA
Citrate
Heparin
P700
P800
Storage Storage duration 0 h
1 h
2 h
4 h
6 h
12 h
24 h
48 h
74 h
96 h
Storage Storage temperature Wet ice
Room temperature
Analyte Extraction and Purification Protease inhibitor Cocktail
No protease inhibitor added
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Study Purpose
The purpose of this paper was to assess the effect of pre-centrifugation storage of whole blood in tubes containing difference anticoagulants with or without protease inhibitors on the levels of active forms of select circulating peptide gut hormones in plasma and non-anticoagulated blood. Blood was collected from at least six healthy volunteers into different tube types, pooled (10 µl per volunteer), spiked with the target peptide, and subjected to a pre-centrifugation delay (0-96 h) on wet ice or at room temperature. Specimens were then centrifuged for 15 min for 2,500 x g and were analyzed immediately or frozen at -80°C.
Summary of Findings:
The peak intensity of G36A declined in response to pre-centrifugation storage of whole blood on wet ice. The calculated half-life of G36A in whole blood anticoagulated with EDTA was 20.6 h and for non-anticoagulated blood was 22.4 h. While the addition of a DPP-IV inhibitor (Diprotin A) to whole blood failed to attenuate the effect of delayed centrifugation on EDTA plasma, whole blood collected in P800 tubes remained stable and had a G36A half-life of >72 h. When the pre-centrifugation delay occurred at room temperature G36A half-lives were shortened to 1.3 h for blood in EDTA tubes, and 9.9 h for blood in P800 tubes.
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Peptide MALDI-TOF MS Peptide Radioimmunoassay Peptide Immunoassay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution P800 tubes
EDTA tubes
Analyte Extraction and Purification Protease inhibitor Cocktail
Diprotin A
Storage Time at room temperature 0-96 h
Storage Storage duration 0-96 h
Storage Storage temperature Room temperature
Wet ice
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated