Simulation study of methemoglobin reduction in erythrocytes

Abstract

In an integrative approach to gaining a better understanding of cell-wide molecular networks at the systems level (systems biology), many attempts at computer simulations of metabolic networks have been made. Because of the simplicity of its structure and components, and the availability of kinetic information, erythrocyte metabolism has been the focus of mathematical modeling for over three decades [1], and is a leading example of the use of mathematical modeling not only for understanding biochemical regulation, but also as the basis for the construction of other mathematical frameworks [2][3][4][5]. For example, models of erythrocyte metabolism were able to predict the importance of the de novo synthesis of glutathione and its mechanism of action in glucose-6-phosphate dehydrogenase (G6PDH)-deficient cells [6], and to analyze Methemoglobin (metHb), an oxidized form of hemoglobin, is unable to bind and carry oxygen. Erythrocytes are continuously subjected to oxidative stress and nitrite exposure, which results in the spontaneous formation of metHb. To avoid the accumulation of metHb, reductive pathways mediated by cytochrome b5 or flavin, coupled with NADH-dependent or NADPH-dependent metHb reductases, respectively, keep the level of metHb in erythrocytes at less than 1% of the total hemoglobin under normal conditions. In this work, a mathematical model has been developed to quantitatively assess the relative contributions of the two major metHbreducing pathways, taking into consideration the supply of NADH and NADPH from central energy metabolism. The results of the simulation experiments suggest that these pathways have different roles in the reduction of metHb; one has a high response rate to hemoglobin oxidation with a limited reducing flux, and the other has a low response rate with a high capacity flux. On the basis of the results of our model, under normal oxidative conditions, the NADPH-dependent system, the physiological role of which to date has been unclear, is predicted to be responsible for most of the reduction of metHb. In contrast, the cytochrome b5-NADH pathway becomes dominant under conditions of excess metHb accumulation, only after the capacity of the flavin-NADPH pathway has reached its limit. We discuss the potential implications of a system designed with two metHbreducing pathways in human erythrocytes.Abbreviations cytb5, cytochrome b5; cytb5R, (NADH-dependent) cytochrome b5 reductase; FR, (NADPH-dependent) flavin reductase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G6PDH, glucose-6-phosphate dehydrogenase; GSSG, oxidized glutathione; GSH, glutathione (reduced form); LDH, lactate dehydrogenase; metHb, methemoglobin.

Reactions of Antimalarial Peroxides with Each of Leucomethylene Blue and Dihydroflavins: Flavin Reductase and the Cofactor Model Exemplified

“…Without Fre, partial reduction of MB by NADPH to a steady-state concentration consisting of 25 % LMB took place; the extent of reduction is about the same as that observed with NADH. [5] Thus, as previously reported, [23,25] MB serves as a substrate for Fre. Treatment of the NADPH-Fre-LMB solution with an excess (10 equiv) of each of artemisinin 1 and tetraoxane 4 resulted in rapid reappearance of the absorption at l max 660 nm as LMB was oxidized to MB ( Figure 2 and figure S2 Supporting Information).…”

“…[25,27] Of interest is their ability to use the acceptors MB or RF administered during treatment of methemoglobinemia. [23,25] In human erythrocytes, MB and RF are reduced by NADPH-flavin reductase to LMB and RFH 2 , which in turn reduce methemoglobin to Hb. The MB and RF thereby regenerated are reduced by the NADPH-flavin reductase in continuation of the cycle.…”

“…The MB and RF thereby regenerated are reduced by the NADPH-flavin reductase in continuation of the cycle. [23,25] In a malaria-infected erythrocyte, it is not known if the parasite uses flavin reductase for maintenance of redox homeostasis. A functionally related ferredoxin-NADP + reductase occurs in the parasite apicoplast, [28] and a chorismate synthase possessing flavin reductase activity has been detected in the parasite cytosol.…”

“…Alternatively, in an aqueous medium the dithionite may have to be decomposed by decreasing the pH, and then re-establishing neutral pH prior to conducting the reactions of the of the artemisinins with the reduced conjugates. [5] NAD(P)H-flavin oxidoreductases do not contain a flavin cofactor, but reduce endogenous flavins that bind to an acceptor site by transferring hydride from NAD(P)H. [23][24][25][26][27] Structures of isoforms that include the acceptor binding site have been elucidated, and the manner in which NAD(P)H reduces the bound acceptor has been discussed. [25,27] Of interest is their ability to use the acceptors MB or RF administered during treatment of methemoglobinemia.…”

“…[23,32] The rate greatly exceeds that of Fe III oxidation of thiols, including reduced glutathione GSH, in a biological medium. [23,32] Therefore, a link to the peroxide cofactor model may be provided by Fe III through its assistance of the antimalarial action of peroxides, not by reacting with them, but rather by accelerating turnover of flavin cofactors which enhances the consumption of NADPH. Thus, in Figure 4.…”