PARENTERAL NUTRITION AND BRANCHED CHAIN AMINO ACIDS: AN IN VIVO AND IN VITRO EVALUATION.

Parenteral nutrition (PN) offers the possibility to increase or to ensure nutrient intake, providing nutrients in sufficient doses to meet the veterinary and human patient’s daily requirements. PN is a complex admixture based on amino acids, dextrose, lipids. Safety issues related to parenteral nutrition formulations have led to the development of guidelines for safe practices. The development of new formulation and the evaluation of the already used solution is the main research in PN practice. International guidelines are quite vague regarding the optimal doses of amino acid to administer to malnourished patients affected by liver desease. A decreased ratio of branched-chain amino acids (BCAA) to aromatic amino acids (AAA) is considered an important pathogenetic factor in hepatic disease. The PN solution are composed by different compound, where the BCAAs are always presents. Long-term oral supplementation with branched chain amino acids (BCAAs) as adjuvant during liver injuries, both in humans and animals, is still not totally clear. Aim of the present study was to determine how amino acids in PN and BCAAs on an in vitro study can preserve liver damage and function. First purpose of this study was to evaluate the use of partial parenteral nutrition (PPN) in dogs, using two different solutions: 11 dogs treated with a normal amino acidic concentration (Medium/Low Protein Level group, Freamine concentration ≤ 25%;1,9-2,2 gr/kg/die amino acids/proteins) as control group and 9 dogs with high-amino acid percentage (High Protein Level group, Freamine > 25%; 3,6-6 gr/kg/die amino acids/proteins) as cases. The evaluation of the two differents solutions is based on the liver damage during the therapy. Results reveal a not statistically significatives differencies for liver damage between High Protein Level group and Medium/Low Protein Level group. It means that an a hight amount of amino acids in PN could not affect and overload the hepatic metabolism, despite carrying out their more advantage effects on body lean mass. The second purpose and study, which is the most importan for us, was to evaluate two differents in vitro models selected: A) hepatic rabbit cells plated in 2D monolayer and B) rabbit hepatic cells cultured onto 3D scaffolds, obtained from decellularized rabbit liver. Both A and B cells were treated with BCAAs. All cells adhered and proliferated, once plated. Moreover, the presence of hepatic progenitors (HPCs), were detected by immuno staining assay and mRNA expression. However, A cells were shown to produce albumin and FVII but quickly entered G0 and arrested growth by day 15. In contrast, cells in B, actively replicated and recellularized ECM rabbit liver, mimicking the original organ cyto-architecture, until day 21. Interestingly, under defined conditions, BCAA supplementation promoted vigorous growth of cell organoid and increased hepatocyte albumin and FVII production and expression. A selective amplification of self-renewing bi-potent HPCs was also observed. These results highlight the role of BCAAs in long term supplementation and support the use of 3D cultures as reliable in vitro models. In conclusion, these finding suggest that an high amino acidic diet with a high BCAAs/AAA ratio coud be a valid therapeutic option in cachectic patients or patient with severe livere diseases.