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Minimizing Postsampling Degradation of Peptides by a Thermal Benchtop Tissue Stabilization Method.

Segerström L, Gustavsson J, Nylander I - Biopreserv Biobank (2016)

Bottom Line: Postmortem metabolism in biological samples entails considerable risk for measurements misrepresentative of true in vivo concentrations.Also, simplified extraction protocols for stabilized tissue were tested.The result is improved diagnostic and research value of the samples with great benefits for basic research and clinical work.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Biosciences, Neuropharmacology, Addiction & Behavior, Uppsala University , Uppsala, Sweden .

ABSTRACT
Enzymatic degradation is a major concern in peptide analysis. Postmortem metabolism in biological samples entails considerable risk for measurements misrepresentative of true in vivo concentrations. It is therefore vital to find reliable, reproducible, and easy-to-use procedures to inhibit enzymatic activity in fresh tissues before subjecting them to qualitative and quantitative analyses. The aim of this study was to test a benchtop thermal stabilization method to optimize measurement of endogenous opioids in brain tissue. Endogenous opioid peptides are generated from precursor proteins through multiple enzymatic steps that include conversion of one bioactive peptide to another, often with a different function. Ex vivo metabolism may, therefore, lead to erroneous functional interpretations. The efficacy of heat stabilization was systematically evaluated in a number of postmortem handling procedures. Dynorphin B (DYNB), Leu-enkephalin-Arg(6) (LARG), and Met-enkephalin-Arg(6)-Phe(7) (MEAP) were measured by radioimmunoassay in rat hypothalamus, striatum (STR), and cingulate cortex (CCX). Also, simplified extraction protocols for stabilized tissue were tested. Stabilization affected all peptide levels to varying degrees compared to those prepared by standard dissection and tissue handling procedures. Stabilization increased DYNB in hypothalamus, but not STR or CCX, whereas LARG generally decreased. MEAP increased in hypothalamus after all stabilization procedures, whereas for STR and CCX, the effect was dependent on the time point for stabilization. The efficacy of stabilization allowed samples to be left for 2 hours in room temperature (20°C) without changes in peptide levels. This study shows that conductive heat transfer is an easy-to-use and efficient procedure for the preservation of the molecular composition in biological samples. Region- and peptide-specific critical steps were identified and stabilization enabled the optimization of tissue handling and opioid peptide analysis. The result is improved diagnostic and research value of the samples with great benefits for basic research and clinical work.

No MeSH data available.


Related in: MedlinePlus

Experimental outline of Part I. (A) Standard procedure for dissection of the brain and tissue collection; (B) the three stabilization procedures (see text); (C) test procedures used to examine changes in peptide levels in samples placed on wet ice for 15 minutes; (D) test procedure to examine the efficiency of the stabilization procedure. CCX, cingulate cortex; STR, striatum.
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f1: Experimental outline of Part I. (A) Standard procedure for dissection of the brain and tissue collection; (B) the three stabilization procedures (see text); (C) test procedures used to examine changes in peptide levels in samples placed on wet ice for 15 minutes; (D) test procedure to examine the efficiency of the stabilization procedure. CCX, cingulate cortex; STR, striatum.

Mentions: The experimental design is depicted in Figure 1A–D. All test procedures were compared to our standard procedure (Fig. 1A), in which the brain is rapidly freed and, after hypothalamus removal, sectioned in a precooled matrix; regions of interest are dissected and rapidly frozen on aluminum-encased dry ice (≈−75°C). Test procedures were designed to answer the following questions:


Minimizing Postsampling Degradation of Peptides by a Thermal Benchtop Tissue Stabilization Method.

Segerström L, Gustavsson J, Nylander I - Biopreserv Biobank (2016)

Experimental outline of Part I. (A) Standard procedure for dissection of the brain and tissue collection; (B) the three stabilization procedures (see text); (C) test procedures used to examine changes in peptide levels in samples placed on wet ice for 15 minutes; (D) test procedure to examine the efficiency of the stabilization procedure. CCX, cingulate cortex; STR, striatum.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834486&req=5

f1: Experimental outline of Part I. (A) Standard procedure for dissection of the brain and tissue collection; (B) the three stabilization procedures (see text); (C) test procedures used to examine changes in peptide levels in samples placed on wet ice for 15 minutes; (D) test procedure to examine the efficiency of the stabilization procedure. CCX, cingulate cortex; STR, striatum.
Mentions: The experimental design is depicted in Figure 1A–D. All test procedures were compared to our standard procedure (Fig. 1A), in which the brain is rapidly freed and, after hypothalamus removal, sectioned in a precooled matrix; regions of interest are dissected and rapidly frozen on aluminum-encased dry ice (≈−75°C). Test procedures were designed to answer the following questions:

Bottom Line: Postmortem metabolism in biological samples entails considerable risk for measurements misrepresentative of true in vivo concentrations.Also, simplified extraction protocols for stabilized tissue were tested.The result is improved diagnostic and research value of the samples with great benefits for basic research and clinical work.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Biosciences, Neuropharmacology, Addiction & Behavior, Uppsala University , Uppsala, Sweden .

ABSTRACT
Enzymatic degradation is a major concern in peptide analysis. Postmortem metabolism in biological samples entails considerable risk for measurements misrepresentative of true in vivo concentrations. It is therefore vital to find reliable, reproducible, and easy-to-use procedures to inhibit enzymatic activity in fresh tissues before subjecting them to qualitative and quantitative analyses. The aim of this study was to test a benchtop thermal stabilization method to optimize measurement of endogenous opioids in brain tissue. Endogenous opioid peptides are generated from precursor proteins through multiple enzymatic steps that include conversion of one bioactive peptide to another, often with a different function. Ex vivo metabolism may, therefore, lead to erroneous functional interpretations. The efficacy of heat stabilization was systematically evaluated in a number of postmortem handling procedures. Dynorphin B (DYNB), Leu-enkephalin-Arg(6) (LARG), and Met-enkephalin-Arg(6)-Phe(7) (MEAP) were measured by radioimmunoassay in rat hypothalamus, striatum (STR), and cingulate cortex (CCX). Also, simplified extraction protocols for stabilized tissue were tested. Stabilization affected all peptide levels to varying degrees compared to those prepared by standard dissection and tissue handling procedures. Stabilization increased DYNB in hypothalamus, but not STR or CCX, whereas LARG generally decreased. MEAP increased in hypothalamus after all stabilization procedures, whereas for STR and CCX, the effect was dependent on the time point for stabilization. The efficacy of stabilization allowed samples to be left for 2 hours in room temperature (20°C) without changes in peptide levels. This study shows that conductive heat transfer is an easy-to-use and efficient procedure for the preservation of the molecular composition in biological samples. Region- and peptide-specific critical steps were identified and stabilization enabled the optimization of tissue handling and opioid peptide analysis. The result is improved diagnostic and research value of the samples with great benefits for basic research and clinical work.

No MeSH data available.


Related in: MedlinePlus