Hidden Antioxidative Functions of Reduced Nicotinamide Adenine Dinucleotide Coexisting with Hemoglobin.

Abstract

Ferrous oxyhemoglobin (HbO2) in red blood cells (RBCs) invariably and slowly autoxidizes to form ferric methemoglobin (metHb). However, the level of metHb is always maintained below 0.5% by intracellular metHb reduction of enzymatic systems with coenzymes, such as reduced nicotinamide adenine dinucleotide (NADH), and by superoxide dismutase (SOD) and catalase (CAT), which eliminate reactive oxygen species. Unquestionably, NADH cannot reduce metHb without the corresponding enzymatic system. Our study, however, demonstrated that a high concentration of NADH (100-fold of normal level, equimolar to HbO2) retarded autoxidation of HbO2 in a highly purified Hb solution with no enzymatic system. Furthermore, an inhibitory effect of NADH on metHb formation was observed with additions of oxidants such as H2O2, NO, and NaNO2. Our mechanism assessment elucidated extremely high pseudo-CAT and pseudo-SOD activities of NADH with coexistence of HbO2, and reactivity of NADH with NO. We prepared a model of RBCs (Hb-vesicles, Hb-V) encapsulating purified HbO2 solution and NADH, but no enzymatic system within liposome. We confirmed the inhibitory effect of NADH on both autoxidation and oxidant-induced metHb formation. In addition, an intravenous administration of these Hb-Vs to rats caused significant retardation of metHb formation by approximately 50% compared to the case without NADH coencapsulation. Based on these results, we elucidated a new role of NADH, that is, antioxidative effect via interaction with Hb, in addition to its classical role as a coenzyme.