P.34 Impact of free amino acids, glycomacropeptide and slow-release protein substitutes on oxidative and inflammatory status in human intestinal Caco-2 cells

Background: The treatment of Phenylketonuria (PKU) involves the daily administration of free amino acids mixtures (L-AAs) that have a different intestinal absorption kinetics than natural proteins. Slowrelease protein substitutes (PS) and glycomacropeptide (GMP) represent two alternative PS for PKU. Oxidative stress and inflammation represent an actual concern in PKU diet. Given the known correlations between oxidative-inflammatory stress and the development of non-communicable diseases (NCDs), the choice of appropriate PS becomes crucial to prevent the onset of NCDs in PKU patients. This works aims to evaluate the impact of GMP and slow-release PS on the oxidative and inflammatory status in vitro and in human intestinal Caco-2 cells, comparing it with that of free L-AAs mixtures. Methods: In a first phase, free L-AAs and slow-release PS were compared; a second phase compared free L-AAs, GMP and a 1:1 mixture of free L-AAs+GMP. The antioxidant activity was assessed by 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) assay and Ferric Reducing Antioxidant Power (FRAP) assay. In human intestinal Caco2 cells, the ability to modulate reactive oxygen species (ROS), lipid peroxidation, and nitric oxide (NO) was evaluated. Lipopolysaccharides (LPS) or hydrogen peroxide (H2O2) were used as stimuli for mimicking the oxidative and inflammatory stress. Furthermore, the production of pro-inflammatory (IL-1β, IL-6, IFN-γ and TNF-α) and anti-inflammatory cytokines (IL-10) was assessed after induction of inflammatory stress with LPS. Results: The use of slow-release, GMP and 1:1 free L-AAs+GMP mixtures significantly reduced the DPPH radical, in contrast to free L-AAs. All samples showed iron-reducing activity in the FRAP assay. In the MTT assay, these mixtures showed a better safety profile than that of free L-AAs. They significantly reduced intracellular ROS production, modulated lipid peroxidation, and restored physiological NO levels after the induction with H2O2. They also reduced LPS-induced proinflammatory cytokines production. Conclusions: Free L-AAs appear to significantly worsen the oxidative and inflammatory state on human Caco-2 cells; this state is restored when cells are treated with slow-release L-AAs, GMP or 1:1 mixtures of free L-AAs+GMP. These results provide important preclinical novelties for the choice of PS for PKU patients to prevent the development of NCDs, known to be related to cellular oxidativeinflammatory stress.