Department of Basic Research on Social Recognition and Memory, Research Center for Child Mental Development, Kanazawa University, Kanazawa, 920-8640, Japan.
Laboratory for Social Brain Studies, Research Institute of Molecular Medicine and Pathobiochemistry, and Department of Biochemistry, Krasnoyarsk State Medical University named after Prof. V. F. Voino-Yasentsky, Krasnoyarsk, 660022, Russia.
Peptides. 2021 Dec;146:170649. doi: 10.1016/j.peptides.2021.170649. Epub 2021 Sep 17.
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.
晚期糖基化终产物受体 (RAGE) 结合催产素 (OT) 并将其从血液转运至大脑。由于 RAGE 敲除 (KO) 小鼠的 OT 结合能力丧失，我们预测与野生型 (WT) 小鼠相比，循环中未结合 (游离) OT 的浓度应该升高。然而，这一假设尚未得到验证。不幸的是，免疫测定中循环游离和结合血浆 OT 动力学的评估尚不清楚，部分原因是血浆蛋白的干扰。放射免疫测定 (RIA) 被认为是克服这一问题的金标准方法，但实施起来更具挑战性；因此，商业上可用的酶联免疫吸附测定 (ELISA) 更为常用。在这里，我们开发了一种预处理方法，在进行 ELISA 之前去除血浆中的干扰成分。与固相萃取血浆样品相比，乙腈蛋白沉淀 (PPT) 方法更可靠，需要的步骤更少，可用于测量游离 OT 浓度。使用 ELISA，PPT 提取的血浆样品在 RAGE KO 小鼠中产生的 OT 浓度高于 WT 小鼠。外周 OT 注射后，WT 小鼠中游离 OT 血浆水平立即升高，然后迅速下降，但 KO 小鼠中仍保持较高水平。这些结果表明，经 PPT 预处理的血浆样品似乎更优越，循环可溶性 RAGE 很可能作为 OT 的缓冲液，这表明 RAGE 具有新的生理功能。
