Suppression of Methemoglobin Formation by Glutathione in a Concentrated Hemoglobin Solution and in a Hemoglobin-Vesicle

Abstract

The suppression of methemoglobin (metHb) formation by glutathione (GSH) is difficult because GSH is oxidized not only by the reduction of metHb, but also by oxygen to generate active oxygen species, such as superoxide (O2−•) and hydrogen peroxide (H2O2), which contribute to metHb formation. An effective nonenzymatic reduction of metHb was achieved at a high concentration of Hb (40 wt%, 2.48 × 10−2 M subunits (1 M = 1 mol dm−3)) because the reduction of metHb was accelerated, and at a low partial pressure of oxygen (pO2 = 40 Torr (1 Torr = 133.322 Pa)) because the oxidation of GSH was effectively suppressed. Hb-vesicles, which encapsulate concentrated Hb (40 wt%, 2.48 × 10−2 M subunits), transport oxygen in the blood stream at relatively low pO2 (110 Torr in artery and 40 Torr in venous, 58 Torr in average). The rate of metHb formation in Hb-vesicles was effectively decreased from 3.8 × 10−7 to 1.6 × 10−7 M s−1 by coencapsulating GSH at 58 Torr.

Physical Properties of Hemoglobin Vesicles as Red Cell Substitutes

“…From our studies on the effect of various nonenzymatic reductants, especially thiols including glutathione, it has been determined that the active oxygen species generated during the oxidation of the reductants by O 2 adversely accelerate the metHb formation (Sakai et al, 1994). Hcy has hitherto showed the best suppressive effect as a reductant due to its relatively slow oxidation rate and high metHb reduction rate.…”

Polymer/Hemoglobin Assemblies: Biodegradable Oxygen Carriers for Artificial Red Blood Cells

“…On the other hand, the steric barrier created by PEG modification on the surface of the phospholipid membrane could restrain the aggregation of HbV, and thus stabilize HbV dispersions 86, 87. The methemoglobin (metHb) level of PEGylated HbV [PEG/ liposome‐encapsulated hemoglobin (LEH)] dispersions could also be suppressed by co‐encapsulation of reductants or catalase, like glutathione (GSH), homocysteine (Hcy) etc 88–92. The co‐encapsulation of GSH in LEH decreased the rate of metHb formation from 3.8 × 10 −7 to 1.6 × 10 −7 M · s −1 at 58 Torr 88.…”

“…The methemoglobin (metHb) level of PEGylated HbV [PEG/ liposome‐encapsulated hemoglobin (LEH)] dispersions could also be suppressed by co‐encapsulation of reductants or catalase, like glutathione (GSH), homocysteine (Hcy) etc 88–92. The co‐encapsulation of GSH in LEH decreased the rate of metHb formation from 3.8 × 10 −7 to 1.6 × 10 −7 M · s −1 at 58 Torr 88. If 5 × 10 −3 M of Hcy was encapsulated in HbV, a 40% lower rate of metHb formation ([metHb] = 23%) than that of the HbV without any Hcy ([metHb] = 35%) was confirmed in vitro at p O 2 of 142 Torr at 37 °C for 24 h 92.…”

Solution to the problems of acellular hemoglobins by encapsulation and the intrinsic issues of hemoglobin vesicles as a molecular assembly

“…Therefore, prevention of metHb formation is necessary. A method exists to preserve deoxygenated Hbs in a liquid state using well‐known intrinsic characteristics of Hb: the Hb oxidation rate in a solution is dependent on the oxygen partial pressure; also, deoxyHb is not autoxidized at ambient temperatures 48 . In the case of HbV, not only the encapsulated Hb but also the capsular structure (liposome) must be physically stabilized to prevent irreversible intervesicular aggregation, fusion and leakage of the encapsulated Hb.…”