A new enzymatic assay to quantify inorganic pyrophosphate in plasma

Abstract

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.

Inorganic Pyrophosphate Plasma Levels Are Decreased in Pseudoxanthoma Elasticum Patients and Heterozygous Carriers but Do Not Correlate with the Genotype or Phenotype

“…Moreover, we performed an internal calibration and noted that the readout values could vary between different samples despite having been spiked with equal amounts of ATP, indicating that unknown factors in the plasma can affect the readout and may be responsible for part of the variation between different studies (Supplementary Table S1). The value of an internal ATP standard to obtain reliable measurement results was also recently shown by Lundkvist et al [30]. We cannot exclude that ethnic factors also affect PPi levels [31][32][33][34][35][36].…”

“…As we used a citrate-based assay, such differences in reduction-both relative and absolute-indicate that the anticoagulant may significantly impact the PPi signal readout, as exemplified by the difference found between CTAD and EDTA by Sanchez-Tévar et al [15]. Such an effect of the anticoagulant was recently illustrated by Lundkvist et al, who showed that the amount of PPi detected depended on the anticoagulant used in the unfiltered plasma, with CTAD and heparin yielding higher concentrations compared to EDTA [30]. Moreover, we performed an internal calibration and noted that the readout values could vary between different samples despite having been spiked with equal amounts of ATP, indicating that unknown factors in the plasma can affect the readout and may be responsible for part of the variation between different studies (Supplementary Table S1).…”

The inorganic pyrophosphatases of microorganisms: a structural and functional review

“…A recent paper described the use of coupled enzymatic reactions using ATP sulfurylase to generate ATP and then quantified it by firefly luciferase activity (quantifying the bioluminescence) and included an internal ATP standard to correct for sample-specific interference since the authors aimed to determine the plasma levels of PPi ( Lundkvist et al, 2023 ). This assay shows excellent precision and accuracy, and interestingly, the anticoagulant EDTA blocked the conversion of ATP into PPi in plasma after blood collection.…”