Skip to main content
SpringerLink
Log in

A new enzymatic assay to quantify inorganic pyrophosphate in plasma

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

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.

Graphical Abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Orriss IR. Extracellular pyrophosphate: the body’s “water softener.” Bone. 2020;134:115243. https://doi.org/10.1016/j.bone.2020.115243.

    Article  CAS  Google Scholar 

  2. Ralph D, van de Wetering K, Uitto J, Li Q. Inorganic pyrophosphate deficiency syndromes and potential treatments for pathologic tissue calcification. Am J Pathol. 2022;192:762–70. https://doi.org/10.1016/j.ajpath.2022.01.012.

    Article  CAS  Google Scholar 

  3. Villa-Bellosta R. ATP-based therapy prevents vascular calcification and extends longevity in a mouse model of Hutchinson-Gilford progeria syndrome. Proc Natl Acad Sci USA. 2019;116:23698–704. https://doi.org/10.1073/pnas.1910972116.

    Article  CAS  Google Scholar 

  4. Lomashvili KA, Narisawa S, Millán JL, O’Neill WC. Vascular calcification is dependent on plasma levels of pyrophosphate. Kidney Int. 2014;85:1–6. https://doi.org/10.1038/ki.2013.521.

    Article  CAS  Google Scholar 

  5. Jansen RS, Küçükosmanoglu A, de Haas M, Sapthu S, Otero JA, Hegman IEM, Bergen AAB, Gorgels TGMF, Borst P, van de Wetering K. ABCC6 prevents ectopic mineralization seen in pseudoxanthoma elasticum by inducing cellular nucleotide release. Proc Natl Acad Sci USA. 2013;110:20206–11. https://doi.org/10.1073/pnas.1319582110.

    Article  CAS  Google Scholar 

  6. Jansen RS, Duijst S, Mahakena S, Sommer D, Szeri F, Váradi A, Plomp A, Bergen AA, Elferink RPJO, Borst P, van de Wetering K. ABCC6-mediated ATP secretion by the liver is the main source of the mineralization inhibitor inorganic pyrophosphate in the systemic circulation-brief report. Arterioscler Thromb Vasc Biol. 2014;34:1985–9. https://doi.org/10.1161/atvbaha.114.304017.

    Article  CAS  Google Scholar 

  7. Szeri F, Lundkvist S, Donnelly S, Engelke UFH, Rhee K, Williams CJ, Sundberg JP, Wevers RA, Tomlinson RE, Jansen RS, van de Wetering K. The membrane protein ANKH is crucial for bone mechanical performance by mediating cellular export of citrate and ATP. PLoS Genet. 2020;16:e1008884. https://doi.org/10.1371/journal.pgen.1008884.

    Article  CAS  Google Scholar 

  8. Szeri F, Niaziorimi F, Donnelly S, Fariha N, Tertyshnaia M, Patel D, Lundkvist S, Wetering K. The mineralization regulator ANKH mediates cellular efflux of ATP, not pyrophosphate. J Bone Miner Res. 2022;37:1024–31. https://doi.org/10.1002/jbmr.4528.

    Article  CAS  Google Scholar 

  9. Whyte MP, Landt M, Ryan LM, Mulivor RA, Henthorn PS, Fedde KN, Mahuren JD, Coburn SP. Alkaline phosphatase: placental and tissue-nonspecific isoenzymes hydrolyze phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5′-phosphate. Substrate accumulation in carriers of hypophosphatasia corrects during pregnancy. J Clin Invest. 1995;95:1440–5. https://doi.org/10.1172/jci117814.

    Article  CAS  Google Scholar 

  10. Jin H, Hilaire CS, Huang Y, Yang D, Dmitrieva NI, Negro A, Schwartzbeck R, Liu Y, Yu Z, Walts A, Davaine J-M, Lee D-Y, Donahue D, Hsu KS, Chen J, Cheng T, Gahl W, Chen G, Boehm M. Increased activity of TNAP compensates for reduced adenosine production and promotes ectopic calcification in the genetic disease ACDC. Sci Signal. 2016;9:ra121. https://doi.org/10.1126/scisignal.aaf9109.

    Article  CAS  Google Scholar 

  11. Hilaire CS, Ziegler SG, Markello TC, Brusco A, Groden C, Gill F, Carlson-Donohoe H, Lederman RJ, Chen MY, Yang D, Siegenthaler MP, Arduino C, Mancini C, Freudenthal B, Stanescu HC, Zdebik AA, Chaganti RK, Nussbaum RL, Kleta R, Gahl WA, Boehm M. NT5E mutations and arterial calcifications. N Engl J Med. 2011;364:432–42. https://doi.org/10.1056/nejmoa0912923.

    Article  Google Scholar 

  12. O’Neill WC, Sigrist MK, McIntyre CW. Plasma pyrophosphate and vascular calcification in chronic kidney disease. Nephrol Dial Transplant. 2009;25:187–91. https://doi.org/10.1093/ndt/gfp362.

    Article  CAS  Google Scholar 

  13. Lioufas NM, Pedagogos E, Hawley CM, Pascoe EM, Elder GJ, Badve SV, Valks A, Toussaint ND, on behalf of the IMPROVE-CKD Investigators. aortic calcification and arterial stiffness burden in a chronic kidney disease cohort with high cardiovascular risk: baseline characteristics of the Impact of Phosphate Reduction On Vascular End-Points in Chronic Kidney Disease Trial. Am J Nephrol. 2020;51:201–15. https://doi.org/10.1159/000505717.

    Article  CAS  Google Scholar 

  14. Kavousi M, Desai CS, Ayers C, Blumenthal RS, Budoff MJ, Mahabadi A-A, Ikram MA, van der Lugt A, Hofman A, Erbel R, Khera A, Geisel MH, Jöckel K-H, Lehmann N, Hoffmann U, O’Donnell CJ, Massaro JM, Liu K, Möhlenkamp S, Ning H, Franco OH, Greenland P. Prevalence and prognostic implications of coronary artery calcification in low-risk women. JAMA. 2016;316:2126–9. https://doi.org/10.1001/jama.2016.17020.

    Article  CAS  Google Scholar 

  15. Ryan LM, Kozin F, Mccarty DJ. Quantification of human plasma inorganic pyrophosphate. Arthritis Rheum. 1979;22:892–5. https://doi.org/10.1002/art.1780220813.

    Article  CAS  Google Scholar 

  16. Bernhard E, Nitschke Y, Khursigara G, Sabbagh Y, Wang Y, Rutsch F. A reference range for plasma levels of inorganic pyrophosphate in children using the ATP sulfurylase method. J Clin Endocrinol Metab. 2021;107:109–18. https://doi.org/10.1210/clinem/dgab615.

    Article  Google Scholar 

  17. Sánchez-Tévar AM, García-Fernández M, Murcia-Casas B, Rioja-Villodres J, Carrillo JL, Camacho M, Gils MV, Sánchez-Chaparro MA, Vanakker O, Valdivielso P. Plasma inorganic pyrophosphate and alkaline phosphatase in patients with pseudoxanthoma elasticum. Ann Transl Med. 2019;7:798. https://doi.org/10.21037/atm.2019.12.73.

    Article  CAS  Google Scholar 

  18. Dedinszki D, Szeri F, Kozák E, Pomozi V, Tőkési N, Mezei TR, Merczel K, Letavernier E, Tang E, Saux OL, Arányi T, van de Wetering K, Váradi A. Oral administration of pyrophosphate inhibits connective tissue calcification. EMBO Mol Med. 2017;9:1463–70. https://doi.org/10.15252/emmm.201707532.

    Article  CAS  Google Scholar 

  19. Li Q, Guo H, Chou DW, Berndt A, Sundberg JP, Uitto J. Mutant Enpp1asj mice as a model for generalized arterial calcification of infancy. Dis Models Mech. 2013;6:1227–35. https://doi.org/10.1242/dmm.012765.

    Article  CAS  Google Scholar 

  20. Li Q, Chou DW, Price TP, Sundberg JP, Uitto J. Genetic modulation of nephrocalcinosis in mouse models of ectopic mineralization: the Abcc6(tm1Jfk) and Enpp1(asj) mutant mice. Lab Invest. 2014;94:623–32. https://doi.org/10.1038/labinvest.2014.52.

    Article  CAS  Google Scholar 

  21. Kiss N, Fésűs L, Bozsányi S, Szeri F, Gils MV, Szabó V, Nagy AI, Hidvégi B, Szipőcs R, Martin L, Vanakker O, Arányi T, Merkely B, Wikonkál NM, Medvecz M. Nonlinear optical microscopy is a novel tool for the analysis of cutaneous alterations in pseudoxanthoma elasticum. Laser Med Sci. 2020;35:1821–30. https://doi.org/10.1007/s10103-020-03027-w.

    Article  Google Scholar 

  22. Johnson JC, Shanoff M, Bass ST, Boezi JA, Hansen RG. An enzymic method for determination of inorganic pyrophosphate and its use as an assay for RNA polymerase. Anal Biochem. 1968;26:137–45.

    Article  CAS  Google Scholar 

  23. Alfrey AC, Solomons CC. Bone pyrophosphate in uremia and its association with extraosseous calcification. J Clin Invest. 1976;57:700–5. https://doi.org/10.1172/jci108327.

    Article  CAS  Google Scholar 

  24. Cartier PH, Thuillier L. Measurement of inorganic pyrophosphate in biological fluids and bone tissues. Anal Biochem. 1974;61:416–28. https://doi.org/10.1016/0003-2697(74)90407-2.

    Article  CAS  Google Scholar 

  25. Russell RG, Edwards NA, Hodgkinson A. Urinary pyrophosphate and urolithiasis. Lancet. 1964;2:1446.

    Article  CAS  Google Scholar 

  26. Gu C, Chen X, Liu H. Portable, quantitative, and sequential monitoring of copper ions and pyrophosphate based on a DNAzyme-Fe3O4 nanosystem and glucometer readout. Anal Bioanal Chem. 2021;413:6941–9. https://doi.org/10.1007/s00216-021-03662-4.

    Article  CAS  Google Scholar 

  27. Kiran S, Khatik R, Schirhagl R. Smart probe for simultaneous detection of copper ion, pyrophosphate, and alkaline phosphatase in vitro and in clinical samples. Anal Bioanal Chem. 2019;411:6475–85. https://doi.org/10.1007/s00216-019-02027-2.

    Article  CAS  Google Scholar 

  28. Cook GA, O’Brien WE, Wood HG, King MT, Veech RL. A rapid, enzymatic assay for the measurement of inorganic pyrophosphate in animal tissues. Anal Biochem. 1978;91:557–65. https://doi.org/10.1016/0003-2697(78)90543-2.

    Article  CAS  Google Scholar 

  29. Lust G, Seegmiller JE. A rapid, enzymatic assay for measurement of inorganic pyrophosphate in biological samples. Clin Chim Acta. 1976;66:241–9.

    Article  CAS  Google Scholar 

  30. McGuire MB, Colman CH, Baghat N, Russell RGG. Radiometric measurement of pyrophosphate in cell cultures. Biochem Soc T. 1980;8:529–30. https://doi.org/10.1042/bst0080529.

    Article  CAS  Google Scholar 

  31. Helenius M, Jalkanen S, Yegutkin GG. Enzyme-coupled assays for simultaneous detection of nanomolar ATP, ADP, AMP, adenosine, inosine and pyrophosphate concentrations in extracellular fluids. Biochim Biophys Acta. 2012;1823:1967–75. https://doi.org/10.1016/j.bbamcr.2012.08.001.

    Article  CAS  Google Scholar 

  32. Tolouian R, Connery SM, O’Neill WC, Gupta A. Using a filtration technique to isolate platelet free plasma for assaying pyrophosphate. Clin Lab. 2012;58:1129–34. https://doi.org/10.7754/clin.lab.2012.111101.

    Article  CAS  Google Scholar 

  33. Li Q, Kingman J, van de Wetering K, Tannouri S, Sundberg JP, Uitto J. Abcc6 knockout rat model highlights the role of liver in PPi homeostasis in pseudoxanthoma elasticum. J Invest Dermatol. 2017;137:1025–32. https://doi.org/10.1016/j.jid.2016.11.042.

    Article  CAS  Google Scholar 

  34. Jansen S, Perrakis A, Ulens C, Winkler C, Andries M, Joosten RP, Acker MV, Luyten FP, Moolenaar WH, Bollen M. Structure of NPP1, an ectonucleotide pyrophosphatase/phosphodiesterase involved in tissue calcification. Structure. 2012;20:1948–59. https://doi.org/10.1016/j.str.2012.09.001.

    Article  CAS  Google Scholar 

  35. Gerrard JM, McNicol A. Platelet storage pool deficiency, leukemia, and myelodysplastic syndromes. Leuk Lymphoma. 2009;8:277–81. https://doi.org/10.3109/10428199209051007.

    Article  Google Scholar 

  36. Borst P, Váradi A, van de Wetering K. PXE, a mysterious inborn error clarified. Trends Biochem Sci. 2019;44:125–40. https://doi.org/10.1016/j.tibs.2018.10.005.

    Article  CAS  Google Scholar 

  37. O’Neill WC, Lomashvili KA, Malluche HH, Faugere M-C, Riser BL. Treatment with pyrophosphate inhibits uremic vascular calcification. Kidney Int. 2010;79:512–7. https://doi.org/10.1038/ki.2010.461.

    Article  CAS  Google Scholar 

  38. Pomozi V, Brampton C, van de Wetering K, Zoll J, Calio B, Pham K, Owens JB, Marh J, Moisyadi S, Váradi A, Martin L, Bauer C, Erdmann J, Aherrahrou Z, Saux OL. Pyrophosphate supplementation prevents chronic and acute calcification in ABCC6-deficient mice. Am J Pathol. 2017;187:1258–72. https://doi.org/10.1016/j.ajpath.2017.02.009.

    Article  CAS  Google Scholar 

  39. Li Q, Huang J, Pinkerton AB, Millán JL, van Zelst BD, Levine MA, Sundberg JP, Uitto J. Inhibition of tissue-nonspecific alkaline phosphatase attenuates ectopic mineralization in the Abcc6-/- mouse model of PXE but not in the Enpp1 mutant mouse models of GACI. J Invest Dermatol. 2018. https://doi.org/10.1016/j.jid.2018.07.030.

    Article  Google Scholar 

  40. Yegutkin GG. Nucleotide- and nucleoside-converting ectoenzymes: important modulators of purinergic signalling cascade. Biochim Biophys Acta. 2008;1783:673–94. https://doi.org/10.1016/j.bbamcr.2008.01.024.

    Article  CAS  Google Scholar 

  41. Lundin A (2014) Optimization of the firefly luciferase reaction for analytical purposes. In: Bioluminescence: fundamentals and applications in biotechnology - Volume 2. Springer, pp 31–62

  42. Silcox DC, McCarty DJ. Measurement of inorganic pyrophosphate in biological fluids. Elevated levels in some patients with osteoarthritis, pseudogout, acromegaly, and uremia. J Clin Invest. 1973;52:1863–70. https://doi.org/10.1172/jci107369.

    Article  CAS  Google Scholar 

  43. Suarez-Moreira E, Hannibal L, Smith CA, Chavez RA, Jacobsen DW, Brasch NE. A simple, convenient method to synthesize cobalamins: synthesis of homocysteinylcobalamin, N-acetylcysteinylcobalamin, 2-N-acetylamino-2-carbomethoxyethanethiolatocobalamin, sulfitocobalamin and nitrocobalamin. Dalton Trans. 2006;5269:5269–77. https://doi.org/10.1039/b610158e.

    Article  CAS  Google Scholar 

  44. Veiga-Lopez A, Sethuraman V, Navasiolava N, Makela B, Olomu I, Long R, van de Wetering K, Martin L, Arányi T, Szeri F. Plasma inorganic pyrophosphate deficiency links multiparity to cardiovascular disease risk. Front Cell Dev Biol. 2020;8:573727. https://doi.org/10.3389/fcell.2020.573727.

    Article  Google Scholar 

  45. Kauffenstein G, Yegutkin GG, Khiati S, Pomozi V, Saux OL, Leftheriotis G, Lenaers G, Henrion D, Martin L. Alteration of extracellular nucleotide metabolism in pseudoxanthoma elasticum. J Invest Dermatol. 2018;138:1862–70. https://doi.org/10.1016/j.jid.2018.02.023.

    Article  CAS  Google Scholar 

  46. Pomozi V, Julian CB, Zoll J, Pham K, Kuo S, Tőkési N, Martin L, Váradi A, Saux OL. Dietary pyrophosphate modulates calcification in a mouse model of pseudoxanthoma elasticum: implication for treatment of patients. J Invest Dermatol. 2019;139:1082–8. https://doi.org/10.1016/j.jid.2018.10.040.

    Article  CAS  Google Scholar 

  47. Verschuere S, Navassiolava N, Martin L, Nevalainen PI, Coucke PJ, Vanakker OM. Reassessment of causality of ABCC6 missense variants associated with pseudoxanthoma elasticum based on Sherloc. Genet Med. 2020;29:205–9. https://doi.org/10.1038/s41436-020-00945-6.

    Article  CAS  Google Scholar 

  48. Müller WEG, Schröder HC, Wang X. Inorganic polyphosphates as storage for and generator of metabolic energy in the extracellular matrix. Chem Rev. 2019;119:12337–74. https://doi.org/10.1021/acs.chemrev.9b00460.

    Article  CAS  Google Scholar 

  49. Krishnamurthy GT, Huebotter RJ, Walsh CF, Taylor JR, Kehr MD, Tubis M, Blahd WH. Kinetics of 99mTc-labeled pyrophosphate and polyphosphate in man. J Nucl Med. 1975;16:109–15.

    CAS  Google Scholar 

Download references

Acknowledgements

We thank Arne Lundin (Biothema, SE) for valuable discussions and are grateful to individuals affected by pseudoxanthoma elasticum (PXE) and their continued support of our research.

Funding

This research was funded by National Institutes of Health, Grant R01AR072695 (K.v.d.W.), U.S. Department of State (Fulbright Visiting Scholar Program), National Research, Development and Innovation Office (OTKA FK131946), Hungarian Academy of Sciences (Bolyai János Fellowship BO/00730/19/8, Mobility grant), ELKH-PoC-2022–023 grant, and the Ministry for Innovation and Technology from the source of the National Research, Development and Innovation Fund (ÚNKP-2022 New National Excellence Program) to F.S. Further funding for this work was provided by PXE International for K.v.d.W. and F.S.

Author information

Authors and Affiliations

  1. 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

    Stefan Lundkvist, Fatemeh Niaziorimi, Flora Szeri & Koen van de Wetering

  2. Department of Chemistry (BMC), Uppsala University, Uppsala, Sweden

    Stefan Lundkvist & Gunnar Johansson

  3. Research Centre for Natural Sciences, Institute of Enzymology, Budapest, Hungary

    Flora Szeri

  4. Department of Biochemistry, Semmelweis University, Budapest, Hungary

    Flora Szeri

  5. PXE International, Damascus, MD, USA

    Matthew Caffet & Sharon F. Terry

  6. Department of Microbiology, Radboud University, Nijmegen, The Netherlands

    Robert S. Jansen

Authors
  1. Stefan Lundkvist
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fatemeh Niaziorimi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Flora Szeri
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Matthew Caffet
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sharon F. Terry
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gunnar Johansson
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Robert S. Jansen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Koen van de Wetering
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Koen van de Wetering.

Ethics declarations

Ethics approval

The Institutional Review Board of Genetic Alliance gave ethical approval for the studies involving blood collection of human participants (protocol number JKVDW001). Animal studies were approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals under approval number 02135–1.

Animal welfare

Experiments were set up in a way to prevent pain and suffering of involved animals.

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 46 kb)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lundkvist, S., Niaziorimi, F., Szeri, F. et al. A new enzymatic assay to quantify inorganic pyrophosphate in plasma. Anal Bioanal Chem 415, 481–492 (2023). https://doi.org/10.1007/s00216-022-04430-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-022-04430-8

Keywords

Search

Navigation