Analytical and Bioanalytical Chemistry ( IF 3.8 ) Pub Date : 2022-11-19 , DOI: 10.1007/s00216-022-04430-8 Stefan Lundkvist 1, 2 , Fatemeh Niaziorimi 1 , Flora Szeri 1, 3, 4 , Matthew Caffet 5 , Sharon F Terry 5 , Gunnar Johansson 2 , Robert S Jansen 6 , Koen van de Wetering 1
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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.
Graphical Abstract
中文翻译：
定量血浆中无机焦磷酸盐的新酶法
无机焦磷酸盐（PPi）是一种重要的细胞外矿化调节剂。低血浆 PPi 浓度是软组织钙化的基础，这种钙化存在于几种罕见的遗传性矿化疾病以及慢性肾病和糖尿病等更常见的疾病中。尽管已知血浆 PPi 稳态失调与多种人类疾病有关，但目前尚无可靠的定量方法。我们在这里描述了一种 PPi 测定，该测定使用 ATP 硫酸化酶将 PPi 转化为 ATP。随后通过基于萤火虫荧光素酶的生物发光对生成的 ATP 进行定量。使用内部 ATP 标准品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。该测定经过验证，显示出添加到人血浆样本中的 PPi 具有出色的精密度 (< 3.5%) 和准确度 (93–106%)。我们发现，在测试的几种抗凝剂中，只有 EDTA 能有效阻止采血后血浆中 ATP 转化为 PPi。此外，通过 300,000 Da 分子量截留膜的过滤降低了血浆 PPi 的变异性，并去除了膜封闭隔室中存在的 ATP（可能是血小板）。应用于野生型和Abcc6 −/−大鼠（一种已建立的 PPi 循环水平较低的动物模型）的血浆样本中，新测定法显示出比我们实验室之前常规使用的测定法更低的变异性。总之，我们在这里报告了一种新的、稳健的测定血浆中 PPi 浓度的方法，由于其高灵敏度、精密度和准确性，该方法优于目前可用的测定方法。
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