SCOHIA PHARMA Inc., 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
SCOHIA PHARMA Inc., 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
J Chromatogr A. 2022 Oct 11;1681:463450. doi: 10.1016/j.chroma.2022.463450. Epub 2022 Aug 27.
Diphosphoinositol pentakisphosphate (InsP) is an inositol pyrophosphate generated by inositol hexakisphosphate kinases (IP6Ks) that regulate diverse biological functions in cells. To date, we have a limited understanding of the InsP biology owing to limited data on InsP levels in blood or other tissues. Given the significant role of InsP in maintaining biological homeostasis, further advancement in InsP measurement is essential. In this study, we report a highly sensitive liquid chromatography with tandem mass spectrometry method for determining InsP levels in whole blood, which is an easily accessible tissue and for which knowledge on InsP levels is limited. We applied a perchloric acid-based method to increase the extraction efficiency of InsP from the cells. Subsequently, we combined a YMC-Triart C18 metal-free column, ion-pair reagents, EDTA, methylenediphosphonic acid, and N,N,N',N'-Ethylenediaminetetrakis(methylenephosphonic acid) to minimize InsP adsorption to the detection system. We prepared blood quality control samples that were highly exposed to IP6K inhibitor, showing minimum InsP levels, to decrease the lower quantification limit of InsP. Furthermore, we applied rat plasma, which was found to show no InsP levels, as a surrogate matrix. This setting resulted in the highly sensitive detection of InsP levels in blood obtained from rats, with a quantification sensitivity of 1 ng of InsP per mL of blood. The current method demonstrated acceptable accuracy (100 ± 15%) and precision (coefficient of variation ≤ 15%) in rat blood. Using this method, we revealed endogenous basal levels of InsP (37.4 ng/mL) in blood obtained from normal rats, and decreased levels of InsP (4.1-21.7 ng/mL) in blood obtained from IP6K inhibitor-administered rats. In summary, we established a highly sensitive method for measuring InsP and revealed its levels in rat blood. The current findings may help in understanding InsP biology.
双磷酸肌醇五磷酸（InsP）是由肌醇六磷酸激酶（IP6K）生成的肌醇焦磷酸，在细胞中调节多种生物学功能。迄今为止，由于血液或其他组织中 InsP 水平的数据有限，我们对 InsP 生物学的了解有限。鉴于 InsP 在维持生物体内平衡中的重要作用，InsP 测量的进一步发展至关重要。在这项研究中，我们报告了一种灵敏的液相色谱-串联质谱法，用于测定全血中的 InsP 水平，全血是一种易于获得的组织，关于其 InsP 水平的知识有限。我们应用基于高氯酸的方法来提高 InsP 从细胞中的提取效率。随后，我们结合使用 YMC-Triart C18 无金属柱、离子对试剂、EDTA、亚甲基二膦酸和 N,N,N',N'-乙二胺四亚甲基膦酸，以最大限度地减少 InsP 对检测系统的吸附。我们制备了高度暴露于 IP6K 抑制剂的血液质控样品，这些样品显示出最低的 InsP 水平，以降低 InsP 的定量下限。此外，我们应用大鼠血浆作为替代基质，发现其不含有 InsP 水平。这种设置使得能够从大鼠血液中高度灵敏地检测 InsP 水平，血液的定量灵敏度为每毫升血液 1ng 的 InsP。该方法在大鼠血液中显示出可接受的准确性（100±15%）和精密度（变异系数≤15%）。使用该方法，我们揭示了正常大鼠血液中内源性基础水平的 InsP（37.4ng/mL），以及给予 IP6K 抑制剂的大鼠血液中 InsP 水平降低（4.1-21.7ng/mL）。总之，我们建立了一种高度灵敏的测量 InsP 的方法，并揭示了其在大鼠血液中的水平。目前的研究结果可能有助于了解 InsP 生物学。
