Department of Dermatology and Cutaneous Biology, Jefferson Institute of Molecular Medicine and PXE International Center of Excellence in Research and Clinical Care, Sidney Kimmel Medical College, Thomas Jefferson University, 233 S 10th Street, PA, 19107, Philadelphia, USA.
Department of Chemistry (BMC), Uppsala University, Uppsala, Sweden.
Anal Bioanal Chem. 2023 Jan;415(3):481-492. doi: 10.1007/s00216-022-04430-8. Epub 2022 Nov 19.
Inorganic pyrophosphate (PPi) is a crucial extracellular mineralization regulator. Low plasma PPi concentrations underlie the soft tissue calcification present in several rare hereditary mineralization disorders as well as in more common conditions like chronic kidney disease and diabetes. Even though deregulated plasma PPi homeostasis is known to be linked to multiple human diseases, there is currently no reliable assay for its quantification. We here describe a PPi assay that employs the enzyme ATP sulfurylase to convert PPi into ATP. Generated ATP is subsequently quantified by firefly luciferase-based bioluminescence. An internal ATP standard was used to correct for sample-specific interference by matrix compounds on firefly luciferase activity. The assay was validated and shows excellent precision (< 3.5%) and accuracy (93-106%) of PPi spiked into human plasma samples. We found that of several anticoagulants tested only EDTA effectively blocked conversion of ATP into PPi in plasma after blood collection. Moreover, filtration over a 300,000-Da molecular weight cut-off membrane reduced variability of plasma PPi and removed ATP present in a membrane-enclosed compartment, possibly platelets. Applied to plasma samples of wild-type and Abcc6 rats, an animal model with established low circulating levels of PPi, the new assay showed lower variability than the assay that was previously in routine use in our laboratory. In conclusion, we here report a new and robust assay to determine PPi concentrations in plasma, which outperforms currently available assays because of its high sensitivity, precision, and accuracy.
无机焦磷酸（PPi）是一种重要的细胞外矿化调节剂。几种罕见的遗传性矿化障碍以及更常见的慢性肾脏病和糖尿病等病症中，存在着低血浆 PPi 浓度的情况，这些病症都伴有软组织钙化。尽管已经知道失调的血浆 PPi 动态平衡与多种人类疾病有关，但目前还没有可靠的方法来定量检测它。在这里，我们描述了一种 PPi 检测方法，该方法利用酶 ATP 硫酸酯酶将 PPi 转化为 ATP。生成的 ATP 随后通过萤火虫荧光素酶的生物发光进行定量。通过使用内部 ATP 标准来校正基质化合物对萤火虫荧光素酶活性的样品特异性干扰，对该检测方法进行了验证，结果显示，该检测方法对人血浆样本中添加的 PPi 具有出色的精密度（<3.5%）和准确性（93-106%）。我们发现，在所测试的几种抗凝剂中，只有 EDTA 能有效阻止血液采集后血浆中 ATP 转化为 PPi。此外，通过 300,000-Da 分子量截止膜过滤可降低血浆 PPi 的变异性，并去除可能存在于膜封闭隔室（如血小板）中的 ATP。将该新检测方法应用于野生型和 Abcc6 大鼠的血浆样本，该动物模型具有已建立的低循环 PPi 水平，与我们实验室之前常规使用的检测方法相比，新检测方法显示出更低的变异性。总之，我们在这里报告了一种新的、强大的检测方法，用于确定血浆中的 PPi 浓度，该方法具有高灵敏度、高精度和准确性，优于目前可用的检测方法。
