AB0935 HYDROLYSIS OF INORGANIC PYROPHOSPHATE IN HUMAN SYNOVIAL FLUID BY IMMOBILIZED PYROPHOSPHATASES
Authors: A. Valueva, R. Romanov, S. Mariasina, M. Elisеev, E. Rodina
Journal: Annals of the Rheumatic Diseases (2020)

Background: Calcium pyrophosphate deposition (CPPD) disease, alternatively known as pseudogout, is a type of arthropathy caused by a deposition of microcrystals of calcium pyrophosphate (CaPPi) in joint tissues, particularly in fibrocartilage and hyaline cartilage. Up to date, specific treatment of CPPD disease has not been developed. Conventional therapy is focused on anti-inflammatory and immunosuppressive treatment aimed at the prevention of acute symptoms. Previously we suggested for this purpose inorganic pyrophosphatases (PPases) immobilized on nanodiamonds of detonation synthesis (NDs) [1]. We synthesized and characterized conjugates of NDs with PPases from Escherichia coli and Mycobacterium tuberculosis (Ec-PPase or Mt-PPase) using amino-substituted NDs with (ND-L) or without linker (ND-NH 2 ). The conjugates showed high enzymatic activity in the in vitro model system simulating the ionic composition of synovial fluid Objectives: The present work is focused on testing enzyme-based agents for the partial dissolution of calcium pyrophosphate deposits in the synovial fluid of patients with CPPD disease. Methods: PPase activity of soluble and immobilized PPases was evaluated as the rate of PPi decrease with time. 1 mM PP i was added to the synovial fluid of patients with CPPD containing the samples of PPases, and the amount of PP i in the samples was determined every 2-4 hours using 31 P NMR spectroscopy. Results: We demonstrated the possibility of differential detection of pyrophosphate (PP i ) among the spectrum of phosphorus-containing compounds in the synovial fluid and used the rate of decrease of exogenous PP i to estimate hydrolytic activity of PPases and PPase-based conjugates. The hydrolysis followed the first order reaction kinetics and the rate constants by different samples of PPases are presented in Table 1. Non-enzymatic hydrolysis of exogenous PP i was quite moderate or not observed at all. Ec-PPase (both soluble and immobilized) showed very low hydrolytic activity with the residual PP i levels above 80%. However, all samples of Mt-PPase showed significant hydrolytic activity. In two cases of three, PP i was almost completely hydrolyzed within 20 hours. The maximum activity (2.24 U•mg -1 ) was shown by Mt-PPase immobilized on ND-L. Hydrolytic activity for all studied enzymes is only 1-2% of such values under optimal in vitro conditions, most probably due to the inhibiting effect of calcium in synovial fluid. Nevertheless, the activity of Mt-PPase-based samples was comparable with the values typical for some classes of hydrolases. It allows us to consider the suggested PPase-based materials as promising agents for the hydrolysis of PP i in the joint tissues in vivo . Table 1. PP i hydrolysis in the synovial fluid by soluble or immobilized PPases. Samples k , hr -1 A , U·mg -1 Control - 0.010 ± 0.001 0 ND-NH 2 0 0 ND-L 0.013 ± 0.002 0 Ес-РРase Soluble 0.025 ± 0.003 0.33 ± 0.04 Immobilized on ND-NH 2 0 0 Immobilized on ND-L 0.017 ± 0.003 0.22 ± 0.04 Mt-PPase Soluble 0.10 ± 0.01 1.3 ± 0.1 Immobilized on ND-NH 2 0.04 ± 0.01 0.53 ± 0.05 Immobilized on ND-L 0.17 ± 0.02 2.2 ± 0.3 Conclusion: In this work, we applied the 31 P NMR spectroscopy to estimate the quantity of P i and PP i in synovial fluids of patients with CPPD disease. The conjugates of bacterial PPases with detonation nanodiamond were demonstrated to retain enzymatic activity in the hydrolysis of exogenous PP i in human synovial fluid. These results provide the basis for the further tests of PPase-based conjugates on animal models in vivo . References: [1]Rodina E.V., Valueva A.V., Yakovlev R.Y., Vorobyeva N.N., Kulakova I.I., Lisichkin G.V., Leonidov N.B. Immobilization of inorganic pyrophosphatase on nanodiamond particles retaining its high enzymatic activity // Biointerphases, Vol. 10, No. 4, 2015. P. 041005. Disclosure of Interests: Anastasiya Valueva: None declared, Roman Romanov: None declared, Sofia Mariasina: None declared, Maxim Elisеev Speakers bureau: Novartis, Menarini Group, Alium, Elena Rodina: None declared