Free-radical-mediated hyaluronan degradation in vitro: effect of N-(2-mercapto-2-methylpropionyl)-L-cysteine

Authors: Mária Baňasová 1    Katarína Valachová 1    Jozef Rychlý 2    Ivica Janigová 2    Katarína Csomorová 2    Vlasta Sasinková 3    Raniero Mendichi 4    Ivo Juránek 1    Ladislav Šoltés 1   
1 Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, SK-84104, Bratislava, Slovakia    2 Polymer Institute, Slovak Academy of Sciences, SK-84541, Bratislava, Slovakia    3 Institute of Chemistry, Slovak Academy of Sciences, SK-84538, Bratislava, Slovakia    4 Istituto per lo Studio delle Macromolecole, Consiglio Nazionale delle Ricerche, I-20133 Milano, Italy   
Year: 2014
Section: Organic, bioorganic and pharmaceutical chemistry, pharmacology
Abstract No.: 1065
ISBN: 978-80-970712-6-4

High-molar-mass hyaluronan (HA) under pathological conditions often degrades yielding low-molar-mass HA fragments. Reactive oxygen species may initiate degradation of high-molar-mass HA leading to a loss of its physiological function. The present study was designed to test the effect of the antirheumatic drug N-(2-mercapto-2-methylpropionyl)-l-cysteine against free-radical-mediated HA degradation initiated by 100-µM ascorbate plus 1-µM Cu(II). Time-dependent decrease of HA dynamic viscosity reflecting HA degradation was recorded by rotational viscometry. Oximetry was applied to follow the overall process. Free-radical scavenging activity of the compound tested was evaluated by the ABTS and DPPH assays. HA fragments were studied by non-isothermal chemiluminometry (CL), thermogravimetry (TGA), differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FT-IR), inductively coupled plasma mass spectrometry (ICP-MS) and size-exclusion chromatography with a multi-angle light scattering photometer (SEC-MALS). Rotational viscometry revealed that N-(2-mercapto-2-methylpropionyl)-l-cysteine completely inhibited the decrease of dynamic viscosity of HA solution, which might indicate N-(2-mercapto-2-methylpropionyl)-l-cysteine-mediated protection of HA from its oxidative degradation. Scavenging activity of N-(2-mercapto-2-methylpropionyl)-l-cysteine represented by IC50 4 and 9 μM for ABTS•+ and DPPH respectively is really valuable. Oximetry showed that compound tested effectively reduced oxygen consumption after initiation of the oxidative HA degradation. On the contrary, the data of SEC-MALS indicated that the oxidative HA degradation took place also in the presence of compound tested. Furthermore, CL, TGA and DSC demonstrated that N-(2-mercapto-2-methylpropionyl)-l-cysteine did not significantly prevent the decrease of hydroperoxide content in the HA sample. Finally, FT-IR and ICP-MS showed that generated thiyl radicals did not incorporate into the HA biopolymer. Concluding, our data suggest that effects of N-(2-mercapto-2-methylpropionyl)-l-cysteine towards oxidative HA degradation observed in vitro could be partially involved in preventing/treating the oxidative degradation of HA in vivo.

Supported by the VEGA grants 2/0011/11, 2/0149/12, APVV 0351-10.