Análise de modificações proteicas induzidas in vitro sobre a formação de produtos proteicos de oxidação avançada e a quantificação de proteína C-reativa

Abstract

Non-enzymatic protein modification reactions occur physiologically and affect many proteins. In some situations, such as kidney and inflammatory diseases, these reactions are exacerbated. The consequences of these modifications affect both the protein structure, leading to loss of function and native characteristics, and induce these molecules to act as inflammatory and chemical signaling mediators. Carbamylation, a reaction observed mainly in patients with chronic kidney disease, demonstrates the accumulation of urea and other toxic products, leading to the addition of a carbamyl group to proteins. Oxidation, in turn, can occur from interaction with a series of agents, but the specific reaction with hypochlorous acid (HOCl) is capable of forming advanced oxidation protein products (AOPP). Both are already established for some proteins, however, there is still no complete understanding of their formation on proteins like gammaglobulins and C-reactive protein (CRP). Thus, the present study aimed to investigate the formation of AOPP from the exposure of albumin and gammaglobulins to HOCl and potassium cyanate (KOCN), a known carbamylating agent, and the susceptibility of CRP in matrix of standard solution and human serum to KOCN and urea, and the impact on their quantification by immunoturbidimetric method, in vitro. Both albumin and gammaglobulins were sensitive to oxidation and AOPP formation against both agents, HOCl and KOCN, concentration-dependent. Interestingly, gamma globulins, despite having been incubated at a lower concentration than albumin, formed a greater amount of AOPP when exposed to HOCl. Also, the formation of AOPP from the reaction of the proteins with KOCN was observed, indicating it as a carbamylating agent with oxidizing potential. The impact of the reaction of KOCN and urea with CRP in the two matrices was also evaluated, resulting in a decrease in its laboratory detection through immunoturbidimetry, an important consequence, since the evaluation of this marker in several pathologies is related to the elevation of its levels. Therefore, the present findings contribute to the understanding of the dynamics of protein modifications on the studied proteins and in the formation of AOPP under these conditions, in the understanding of laboratory interferences in the immunoturbidimetric quantification of CRP, and in the participation of the protein modifications studied in the pathophysiology of chronic diseases.