Ammonium removal by erythrocyte-bioreactors based on glutamate dehydrogenase from Proteus sp. jointly with porcine heart alanine aminotransferase.

Excessive ammonium blood concentration causes many serious neurological complications. The medications currently used are not very effective. To remove ammonium from the blood, erythrocyte-bioreactors containing enzymes that processing ammonium have been proposed. The most promising bioreactor contained co-encapsulated glutamate dehydrogenase (GDH) and alanine aminotransferase (ALT). However, a low encapsulation of a commonly used bovine liver GDH (due to high aggregation), makes clinical use of such bioreactors impossible. In this study, new bioreactors containing ALT and non-aggregating GDH at higher loading were first produced using the flow dialysis method and the new bacterial GDH enzyme from Proteus sp. The efficacy of these erythrocyte-bioreactors and their properties (hemolysis, osmotic fragility, intracellular and extracellular activity of included enzymes, erythrocyte indices, and filterability) were studied and compared with native cells during 1-week storage. The ammonium removal rate in vitro by such erythrocyte-bioreactors increased linearly with an increase in encapsulated GDH activity. Alanine in vitro increased in accordance with ammonium consumption, which indicated the joint functioning of both included enzymes. Thus, novel bioreactors for ammonium removal containing GDH from Proteus sp. are promising for clinical use, since they have a more efficient GDH encapsulation and their properties are not inferior to previously obtained erythrocyte-bioreactors.

Theoretical Analysis of the Built-in Metabolic Pathway Effect on the Metabolism of Erythrocyte-Bioreactors That Neutralize Ammonium.

The limitations of the efficiency of ammonium-neutralizing erythrocyte-bioreactors based on glutamate dehydrogenase and alanine aminotransferase reactions were analyzed using a mathematical model. At low pyruvate concentrations in the external medium (below about 0.3 mM), the main limiting factor is the rate of pyruvate influx into the erythrocyte from the outside, and at higher concentrations, it is the disappearance of a steady state in glycolysis if the rate of ammonium processing is higher than the critical value (about 12 mM/h). This rate corresponds to different values of glutamate dehydrogenase activity at different concentrations of pyruvate in plasma. Oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) by glutamate dehydrogenase decreases the fraction of NADPH in the constant pool of nicotinamide adenine dinucleotide phosphates (NADP + NADPH). This, in turn, activates the pentose phosphate pathway, where NADP reduces to NADPH. Due to the increase in flux through the pentose phosphate pathway, stabilization of the ATP concentration becomes impossible; its value increases until almost the entire pool of adenylates transforms into the ATP form. As the pool of adenylates is constant, the ADP concentration decreases dramatically. This slows the pyruvate kinase reaction, leading to the disappearance of the steady state in glycolysis.

Erythrocytes as Carriers: From Drug Delivery to Biosensors.

Drug delivery using natural biological carriers, especially erythrocytes, is a rapidly developing field. Such erythrocytes can act as carriers that prolong the drug's action due to its gradual release from the carrier; as bioreactors with encapsulated enzymes performing the necessary reactions, while remaining inaccessible to the immune system and plasma proteases; or as a tool for targeted drug delivery to target organs, primarily to cells of the reticuloendothelial system, liver and spleen. To date, erythrocytes have been studied as carriers for a wide range of drugs, such as enzymes, antibiotics, anti-inflammatory, antiviral drugs, etc., and for diagnostic purposes (e.g. magnetic resonance imaging). The review focuses only on drugs loaded inside erythrocytes, defines the main lines of research for erythrocytes with bioactive substances, as well as the advantages and limitations of their application. Particular attention is paid to in vivo studies, opening-up the potential for the clinical use of drugs encapsulated into erythrocytes.