Encapsulation of Concentrated Hemoglobin Solution in Phospholipid Vesicles Retards the Reaction with NO, but Not CO, by Intracellular Diffusion Barrier

Abstract

One physiological significance of the red blood cell (RBC) structure is that NO binding of Hb is retarded by encapsulation with the cell membrane. To clarify the mechanism, we analyzed Hb-vesicles (HbVs) with different intracellular Hb concentrations, [Hb] ) and many enzymes; and (iv) modulation of entrapment of endogenous gaseous messenger molecules (NO and CO) (6, 7) because it has been clarified in pathological conditions with hemolysis (8) and in the development of some Hbbased oxygen carriers (HBOCs) (9 -16) that entrapment of endothelium-derived NO induces vasoconstriction, hypertension, reduced blood flow, and vascular damage. CO is also a vasorelaxation factor, especially in hepatic microcirculation (17). Entrapment of CO by a cell-free Hb solution induces constriction of sinusoidal capillaries (18). These side effects of molecular Hb imply the importance of the cellular structure of RBC. Despite such a background in this field, the mechanism of retardation of NO binding by Hb encapsulation in RBC remains controversial (19 -21). It remains unclear whether (i) an unstirred layer is formed as an extracellular diffusion barrier surrounding the RBC (6, 9); (ii) a protein-rich RBC cytoskeletal submembrane becomes a physical barrier against NO diffusion (22, 23); or (iii) gas diffusion is retarded because of the viscous Hb solution in RBC (2). As chemists, it seems to us that these controversies are attributable to the complex and fragile structure of RBC and chemically unstable NO, which make it difficult to analyze the binding rate constant of NO to RBC.Hemoglobin-vesicles (HbVs) or liposome-encapsulated Hbs have been developed as transfusion alternatives. Their efficacy as O 2 carriers is comparable with that of RBC (24 -28). It has been thought that liposomes as a molecular assembly are a fragile capsule. However, appropriate lipid composition and polyethylene glycol modification on the surface of vesicles stabilize the dispersion state (29) and enable stopped-flow measure- * This work was supported in part by Health and Labor Sciences ResearchGrants (Research on Regulatory Science of Pharmaceuticals and Medical Devices), Ministry of Health, Labor, and Welfare, Japan (to H. S. and E. T.) and by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science B16300162 (to H. S.) and 18500368 (to S. T.), and Global COE "Practical Chemical Wisdom" (to S. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Mechanisms of Slower Nitric Oxide Uptake by Red Blood Cells and Other Hemoglobin-containing Vesicles

“…Investigating the reaction kinetics of these vesicles with NO is therefore important to validate their functionality in place of red blood cells. Phospholipid vesicles of differing sizes and concentrations of intracellular hemoglobin may be made (21). Measuring the dependence of their uptake rates of NO on size and hemoglobin concentration may also provide insight into the relative importance of factors intrinsic to the erythrocyte that influence its scavenging rate of NO.…”

“…In their simulations the authors did not include either extracellular diffusion of NO (i.e. extravesicular, or diffusion to a vesicle through the bulk fluid up to (but not through) the membrane) or membrane permeability to NO (21). Only the diffusion of NO inside of a vesicle was simulated.…”

“…The difference in the scavenging rates has been attributed to four possible factors: 1) a red blood cell (RBC)-free zone adjacent to the endothelium due to the velocity gradient in laminar flow (2-4), 2) NO uptake being rate-limited by diffusion of NO to the RBC, which contributes to the phenomenon of an unstirred layer around the RBC (1, 6, 14 -16), 3) an intrinsic, physical RBC membrane barrier to NO diffusion (14,(17)(18)(19)(20), and 4) NO uptake being rate-limited by diffusion of NO within the RBC (intracellular diffusion) (17,21). Although there is no disagreement about the effect of the RBC-free zone along the endothelium on reducing NO reaction rates with red cell hemoglobin, the influence of the unstirred layer versus membrane permeability versus intracellular diffusion of NO is still disputed (21)(22)(23)(24). There is, however, increasing evidence that the membrane of the RBC does provide some resistance to the passage of NO (14,19,20,22).…”

“…To fit all of the experimental and computational data presented in the recent work on phospholipid vesicles (21), we have also performed some simulations with vesicle diameters of 100, 200, 250, 1000, and 2000 nm. The experimental and simulated data in the recent work on phospholipid vesicles by Sakai et al (21) have been obtained by precisely estimating the values from the graphs of their publication to compare it to our own. Some values explicitly given in the publication were also estimated from the graphs and these deviated by a maximum of 1.4% from the exact values.…”

“…In the intracellular region we have used the diffusion constants for both NO and hemoglobin cited in the work on phospholipid vesicles (21), which increase with decreasing concentration of hemoglobin inside a vesicle. The total hemoglobin (in heme) used in our simulations was 1.5 M and the initial concentration of NO in the whole reaction volume was 1.9 M, as in the publication on vesicles (21 (Table 1). For simulations where intracellular diffusion was varied while extracellular diffusion and membrane permeability were constant the NO and hemoglobin diffusion coefficients were both multiplied by the same factor.…”