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.

Keywords: ATP sulfurylase; Ectopic mineralization; Mineralization inhibitor; Plasma; Pyrophosphate; Vascular calcification.

Figure 1.. Concept of quantification of PPi using ATP sulfurylase (ATPS).

(A) enzymatic reaction used to convert PPi into ATP. (B) Schematic overview of the assay to quantify ATP and PPi in plasma. In cuvette 1, which contains a reaction mixture with luciferase and luciferin, the ATP concentration of the sample is determined. In cuvette 2, which contains a reaction mixture with ATPS, APS, luciferase and luciferin, the combined concentration of ATP and PPi is determined. The PPi concentration is subsequently calculated by subtracting the ATP concentration quantified in cuvette 1 from the combined ATP and PPi concentration quantified in cuvette 2. (C) Example of the assay showing how 5 μl of a 1 μM PPi standard (arrow) was quantitatively converted into ATP. After addition of the 10 μM ATP standard the PPi concentration is calculated from the ratio in increase of luminescence induced by addition of PPi and ATP, respectively. Luminescence is continuously followed in a luminometer. PPi and ATP were added after shortly opening the luminometer. Notably, to add sample as well as ATP standard the luminometer is opened, which automatically stops data acquisition. This explains the absence of signal in panel C at the time the sample or ATP standard is added. RLU: Relative Light Units. Panels B and C were created with BioRender.com.

Figure 2.. Linearity of luminescence induced by addition PPi and ATP and application to a human plasma sample.

Increasing concentrations of ATP (A) or PPi (B) were added to the reaction mixture, generating signals that were highly linear with concentration. In panels C and D, the concentration of ATP and of ATP + PPi together were determined in a filtered plasma sample containing CTAD and EDTA as anticoagulants. The PPi concentration was calculated by subtracting the ATP concentration (panel C) from the combined ATP and PPi concentration (panel D). Of note, the first data points after addition of ATP (red arrowheads) are lower due to the shortened integration time after closing the luminometer. RLU: Relative Light Units. In panels A and B, the SD is smaller than the size of the symbols. The data underlying panels A and B are presented in the supplemental data section in tables S1 and S2, respectively.

Figure 3.. Blood processing affects the concentrations of ATP and PPi detected in plasma.

Blood was collected from 3 participants via puncture of the antecubital vein and uncoagulated using the indicated anticoagulants. After preparation of plasma, ATP (A) and PPi (B) were determined in plasma not further processed (“STD”), in plasma that was filtered over a 300,000 mwco filter (“filtered”) and in platelet-rich plasma (“platelet-rich”). Samples were diluted 1:5 in TE buffer before analysis. CTAD: citrate, theophylline, adenosine dipyridamole. EDTA: ethylenediaminetetraacetic acid. Values represent single determinations.

Figure 4.. Stability of PPi and ATP in plasma depends on the used anticoagulant.

Filtered plasma of participant 3, prepared using the indicated anticoagulants, was stored at room temperature (22 °C) and ATP (A) and PPi (B) were quantified at the indicated days in samples diluted 1:5 in TE buffer. Of note, ATP was below the limit of detection in heparin plasma (see Figure 3B). Data represent single determinations performed on the indicated day after the start of storage at room temperature.

Figure 5.. Comparison of the performance of the new (A) and old (B) ATPS-based PPi assay on plasma samples collected from wild type (WT) and Abcc6−/− rats.

Blood was collected from rats under terminal isoflurane anaesthesia by cardiac puncture in CTAD vacuum tubes. Directly after collection 50 μL of a 15% EDTA solution was added, and filtered plasma was prepared as detailed in the materials and methods section. PPi concentrations were determined in the resulting filtered plasma using the old and new assay. Each group contained 2 male and 2 female animals. Data represent individual data points and mean.