Red Blood Cells Donate Electrons to Methylene Blue Mediated Chemical Reduction of Methemoglobin Compartmentalized in Liposomes in Blood

Abstract

Electron-energy-rich coenzymes in cells, NADH and NADPH, are re-energized repeatedly through the Embden-Meyerhof and pentose-phosphate glycolytic pathways, respectively. This study demonstrates extraction of their electron energies in red blood cells (RBCs) for in vivo extracellular chemical reactions using an electron mediator shuttling across the biomembrane. Hemoglobin-vesicles (HbVs) are an artificial oxygen carrier encapsulating purified and concentrated Hb solution in liposomes. Because of the absence of a metHb-reducing enzymatic system in HbV, HbO2 gradually autoxidizes to form metHb. Wistar rats received HbV suspension (10 mL/kg body weight) intravenously. At the metHb level of around 50%, methylene blue [MB(+); 3,7-bis(dimethylamino)phenothiazinium chloride] was injected. The level of metHb quickly decreased to around 16% in 40 min, remaining for more than 5 h. In vitro mixing of HbV/MB(+) with RBCs recreated the in vivo metHb reduction, but not with plasma. NAD(P)H levels in RBCs decreased after metHb reduction. The addition of glucose facilitated metHb reduction. Liposome-encapsulated NAD(P)H, a model of RBC, reduced metHb in HbV in the presence of MB(+). These results indicate that (i) NAD(P)H in RBCs reacts with MB(+) to convert it to leukomethylene blue (MBH); (ii) MB(+) and MBH shuttle freely between RBC and HbV across the hydrophobic lipid membranes; and (iii) MBH is transferred into HbV and reduces metHb in HbV. Four other electron mediators with appropriate redox potentials appeared to be as effective as MB(+) was, indicating the possibility for further optimization of electron mediators. We established an indirect enzymatic metHb reducing system for HbV using unlimited endogenous electrons created in RBCs in combination with an effective electron mediator that prolongs the functional lifespan of HbV in blood circulation.

In vivoevaluation of electron mediators for the reduction of methemoglobin encapsulated in liposomes using electron energies produced by red blood cell glycolysis

“…Anesthetized healthy male Wistar rats received HbV with about 50% metHb level; then they received a dye solution. An earlier study confirmed that the reduction efficiency of MB increased with a larger injection amount in the range of 0.42-1.27 mg/kg body weight [16]. Assuming that the other dyes show similar profiles, the injection amount was fixed to 1.27 mg MB/kg body weight in this study, which is equal to approximately 4 lmol/kg body weight.…”

“…After 5 min, 2.6 mM of the dye solution was injected at the dose rate of 1.53 ml dye/kg body weight. An earlier study confirmed that an increased dose of MB solution (2.6 mM) in the range 0.506-1.53 ml/kg body weight caused the most effective reduction of metHb [16]. This dosage is below the recommended maximum dosage for MB (2 mg/kg, which is approximately 2.4 ml dye/kg rat [2.6 mM]) for injection in treatment of methemoglobinemia [15].…”

“…The HbV suspension used for this study was prepared according to earlier reported procedures [16]. Outdated human blood was provided by the Japanese Red Cross Society (Tokyo, Japan), from which Hb was purified.…”

“…Because the reduced MBH has a neutral charge and since it has a hydrophobic character, this molecule can readily diffuse through the phospholipid bilayer of both RBCs and HbVs. MBH entering the HbV functions as an electron mediator between NADH/NADPH in RBCs and metHb compartmentalized inside the vesicles [16]. Earlier studies have verified that other cationic organic dyes (phenothiazines, phenazines, phenoxazines, indamine) are also useful in such redox reactions, and are thereby able to participate in the reduction of metHb [17].…”

Development of Time-course Oxygen Binding Analysis for Hemoglobin-based Oxygen Carriers

“…[4][5][6][7] Especially, for the chronic blood shortage all over the world, 8 developing blood substitutes is in urgent demand, and various hemoglobin-based oxygen carriers (HBOCs) have been synthesized. In developing HBOCs, one of the most important properties is its oxygen binding behavior.…”