Ab0935 hydrolysis of Inorganic Pyrophosphate in Human Synovial Fluid by Immobilized Pyrophosphatases

Abstract

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 fromEscherichia coliandMycobacterium tuberculosis(Ec-PPase or Mt-PPase) using amino-substituted NDs with (ND-L) or without linker (ND-NH2). The conjugates showed high enzymatic activity in thein vitromodel system simulating the ionic composition of synovial fluidObjectives: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 PPiwas added to the synovial fluid of patients with CPPD containing the samples of PPases, and the amount of PPiin the samples was determined every 2-4 hours using31P NMR spectroscopy.Results:We demonstrated the possibility of differential detection of pyrophosphate (PPi) among the spectrum of phosphorus-containing compounds in the synovial fluid and used the rate of decrease of exogenous PPito 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 PPiwas quite moderate or not observed at all. Ec-PPase (both soluble and immobilized) showed very low hydrolytic activity with the residual PPilevels above 80%. However, all samples of Mt-PPase showed significant hydrolytic activity. In two cases of three, PPiwas 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 optimalin vitroconditions, 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 PPiin the joint tissuesin vivo.Table 1.PPihydrolysis in the synovial fluid by soluble or immobilized PPases.Samplesk, hr-1A, U·mg-1Control-0.010 ± 0.0010ND-NH200ND-L0.013 ± 0.0020Ес-РРaseSoluble0.025 ± 0.0030.33 ± 0.04Immobilized on ND-NH200Immobilized on ND-L0.017 ± 0.0030.22 ± 0.04Mt-PPaseSoluble0.10 ± 0.011.3 ± 0.1Immobilized on ND-NH20.04 ± 0.010.53 ± 0.05Immobilized on ND-L0.17 ± 0.022.2 ± 0.3Conclusion:In this work, we applied the31P NMR spectroscopy to estimate the quantity of Piand PPiin 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 PPiin human synovial fluid. These results provide the basis for the further tests of PPase-based conjugates on animal modelsin 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