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Stability of glucagon-like peptide 1 and glucagon in human plasma.

Wewer Albrechtsen NJ, Bak MJ, Hartmann B, Christensen LW, Kuhre RE, Deacon CF, Holst JJ - Endocr Connect (2015)

Bottom Line: Surprisingly, the recovery of glucagon was reduced by almost 50% by freezing compared with immediate analysis, regardless of storage time.Plasma handling procedures can significantly influence results of subsequent hormone analysis.Freeze-thaw cycles did not significantly affect stability of GLP-1 or glucagon.

View Article: PubMed Central - PubMed

Affiliation: NNF Center for Basic Metabolic Research and Department of Biomedical SciencesDepartment of ScienceFaculty of Health Science, University of Copenhagen, Blegdamsvej 3B, 12.2, DK-2200 Copenhagen N, Denmark.

No MeSH data available.


The impact of long-term storage at −20 °C (black circle) and at −80 °C (black square), respectively, on the recovery of added (40 pmol/l) GLP-1 and glucagon in human plasma. (A) 7–36NH2 GLP-1 isoform spiked in human plasma and measured with a sandwich ELISA specific for intact GLP-1. (B) Glucagon spiked in human plasma and measured with a C-terminal-specific RIA. (C) Glucagon spiked in human plasma and measured with a N-terminal-specific RIA. Each result represents mean±s.d. of eight replicated determinations of GLP-1 and glucagon measured with different assays and normalized to result from samples extracted immediately after addition. *P<0.05 tested by one-way ANOVA for repeated measurement with post hoc Bonferroni correction. 10 pmol/l data not shown but was similar. Standard curve CV ranging from 6 to 13%.
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fig4: The impact of long-term storage at −20 °C (black circle) and at −80 °C (black square), respectively, on the recovery of added (40 pmol/l) GLP-1 and glucagon in human plasma. (A) 7–36NH2 GLP-1 isoform spiked in human plasma and measured with a sandwich ELISA specific for intact GLP-1. (B) Glucagon spiked in human plasma and measured with a C-terminal-specific RIA. (C) Glucagon spiked in human plasma and measured with a N-terminal-specific RIA. Each result represents mean±s.d. of eight replicated determinations of GLP-1 and glucagon measured with different assays and normalized to result from samples extracted immediately after addition. *P<0.05 tested by one-way ANOVA for repeated measurement with post hoc Bonferroni correction. 10 pmol/l data not shown but was similar. Standard curve CV ranging from 6 to 13%.

Mentions: Intact GLP-1 appeared stable over long-term storage, with concentrations remaining unchanged for up to 12 months, independent of storage temperature (−80 compared with −20 °C; Fig. 4A). Similar results were seen for 9–36NH2 (data not shown). In contrast, glucagon concentrations were significantly (P=0.0003) reduced by long-term storage at both −20 and −80 °C. Recovery, measured with both C- and N-terminal assays dropped by almost half after 1 month, continuing to fall more slowly over the subsequent 5 months before stabilizing for the remainder of the 12 months of the study (Fig. 4B and C). The results were surprising and therefore we repeated the experiment using a new plasma pool to investigate at which time the loss in recovery of glucagon occurs. As shown in Supplementary Figure 2A and B, the recovery of glucagon in human plasma was significantly (P=0.0004) reduced, compared with results of immediate extraction and measurement (but no freezing at all), after 3 h of freezing, whereas the recovery in buffer seemed unaffected for up to 72 h. Similar findings were obtained using C- and N-terminal assays.


Stability of glucagon-like peptide 1 and glucagon in human plasma.

Wewer Albrechtsen NJ, Bak MJ, Hartmann B, Christensen LW, Kuhre RE, Deacon CF, Holst JJ - Endocr Connect (2015)

The impact of long-term storage at −20 °C (black circle) and at −80 °C (black square), respectively, on the recovery of added (40 pmol/l) GLP-1 and glucagon in human plasma. (A) 7–36NH2 GLP-1 isoform spiked in human plasma and measured with a sandwich ELISA specific for intact GLP-1. (B) Glucagon spiked in human plasma and measured with a C-terminal-specific RIA. (C) Glucagon spiked in human plasma and measured with a N-terminal-specific RIA. Each result represents mean±s.d. of eight replicated determinations of GLP-1 and glucagon measured with different assays and normalized to result from samples extracted immediately after addition. *P<0.05 tested by one-way ANOVA for repeated measurement with post hoc Bonferroni correction. 10 pmol/l data not shown but was similar. Standard curve CV ranging from 6 to 13%.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317691&req=5

fig4: The impact of long-term storage at −20 °C (black circle) and at −80 °C (black square), respectively, on the recovery of added (40 pmol/l) GLP-1 and glucagon in human plasma. (A) 7–36NH2 GLP-1 isoform spiked in human plasma and measured with a sandwich ELISA specific for intact GLP-1. (B) Glucagon spiked in human plasma and measured with a C-terminal-specific RIA. (C) Glucagon spiked in human plasma and measured with a N-terminal-specific RIA. Each result represents mean±s.d. of eight replicated determinations of GLP-1 and glucagon measured with different assays and normalized to result from samples extracted immediately after addition. *P<0.05 tested by one-way ANOVA for repeated measurement with post hoc Bonferroni correction. 10 pmol/l data not shown but was similar. Standard curve CV ranging from 6 to 13%.
Mentions: Intact GLP-1 appeared stable over long-term storage, with concentrations remaining unchanged for up to 12 months, independent of storage temperature (−80 compared with −20 °C; Fig. 4A). Similar results were seen for 9–36NH2 (data not shown). In contrast, glucagon concentrations were significantly (P=0.0003) reduced by long-term storage at both −20 and −80 °C. Recovery, measured with both C- and N-terminal assays dropped by almost half after 1 month, continuing to fall more slowly over the subsequent 5 months before stabilizing for the remainder of the 12 months of the study (Fig. 4B and C). The results were surprising and therefore we repeated the experiment using a new plasma pool to investigate at which time the loss in recovery of glucagon occurs. As shown in Supplementary Figure 2A and B, the recovery of glucagon in human plasma was significantly (P=0.0004) reduced, compared with results of immediate extraction and measurement (but no freezing at all), after 3 h of freezing, whereas the recovery in buffer seemed unaffected for up to 72 h. Similar findings were obtained using C- and N-terminal assays.

Bottom Line: Surprisingly, the recovery of glucagon was reduced by almost 50% by freezing compared with immediate analysis, regardless of storage time.Plasma handling procedures can significantly influence results of subsequent hormone analysis.Freeze-thaw cycles did not significantly affect stability of GLP-1 or glucagon.

View Article: PubMed Central - PubMed

Affiliation: NNF Center for Basic Metabolic Research and Department of Biomedical SciencesDepartment of ScienceFaculty of Health Science, University of Copenhagen, Blegdamsvej 3B, 12.2, DK-2200 Copenhagen N, Denmark.

No MeSH data available.