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An improved sample extraction method reveals that plasma receptor for advanced glycation end-products (RAGE)
Stanislav M Cherepanov1, Maria Gerasimenko1, Teruko Yuhi1
1Department of Basic Research on Social Recognition and Memory, Research Center for Child Mental Development, Kanazawa University, Kanazawa, 920-8640, Japan.
Abstract:
The receptor for advanced glycation end-products (RAGE) binds oxytocin (OT) and transports it from the blood to the brain. As RAGE's OT-binding capacity was lost in RAGE knockout (KO) mice, we predicted that circulating concentrations of unbound (free) OT should be elevated compared to wild-type (WT) mice. However, this hypothesis has not yet been investigated. Unfortunately, the evaluation of the dynamics of circulating free and bound plasma OT is unclear in immunoassays, in part because of interference from plasma proteins. A radioimmunoassay (RIA) is considered the gold standard method for overcoming this issue, but is more challenging to implement; thus, commercially available enzyme-linked immunosorbent assays (ELISAs) are more commonly used. Here, we developed a pre-treatment method to remove the interference-causing components from plasma before performing ELISA. The acetonitrile protein precipitation (PPT) approach was reliable, with fewer steps needed to measure free OT concentrations than by solid-phase extraction of plasma samples. PPT-extracted plasma samples yielded higher concentrations of OT in RAGE KO mice than in WT mice using ELISA. After peripheral OT injection, free OT plasma levels spiked immediately then rapidly declined in WT mice, but remained high in KO mice. These results suggest that plasma samples with PPT pre-treatment appear to be superior and that circulating soluble RAGE can most likely serve as a buffer for plasma OT, which indicates a novel physiological function of RAGE.
Insights
Receptor for advanced glycation end-products (RAGE) binds and transports oxytocin (OT). RAGE knockout mice show elevated free OT, suggesting RAGE acts as a plasma OT buffer, a novel physiological role.
Area of Science:
- Neuroendocrinology
- Molecular Biology
- Biochemistry
Background:
- The receptor for advanced glycation end-products (RAGE) is known to bind oxytocin (OT).
- RAGE facilitates OT transport from the blood to the brain.
- The physiological implications of RAGE's OT-binding capacity, particularly in RAGE knockout (KO) models, remain under-investigated.
Purpose of the Study:
- To investigate the hypothesis that RAGE knockout (KO) mice exhibit elevated circulating concentrations of unbound (free) oxytocin (OT).
- To develop and validate a reliable method for measuring free OT in plasma, overcoming interference from plasma proteins.
- To elucidate the role of RAGE in regulating circulating OT levels and its potential buffering function.
Main Methods:
- Development of an acetonitrile protein precipitation (PPT) pre-treatment method for plasma samples prior to ELISA.
- Comparison of PPT method with solid-phase extraction for free OT measurement.
- Quantification of free OT concentrations in wild-type (WT) and RAGE KO mice using ELISA.
- Analysis of free OT dynamics in plasma after peripheral OT injection in WT and RAGE KO mice.
Main Results:
- The PPT pre-treatment method proved reliable and efficient for measuring free OT concentrations.
- RAGE KO mice exhibited significantly higher concentrations of free OT compared to WT mice.
- Following OT injection, free OT levels remained elevated for longer in RAGE KO mice compared to WT mice.
Conclusions:
- The developed PPT pre-treatment method enhances the accuracy of free OT measurement via ELISA.
- Circulating soluble RAGE likely functions as a buffer for plasma OT.
- This study reveals a novel physiological role for RAGE in regulating oxytocin homeostasis.
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