A Novel Hemoglobin-Adenosine-Glutathione Based Blood Substitute: Evaluation of Its Effects on Human Blood Ex Vivo

Abstract

Chemically modified hemoglobin (Hb) solutions are under current investigation as potential red cell substitutes. Researchers at Texas Tech University have developed a novel free Hb based blood substitute product. This blood substitute is composed of purified bovine Hb cross-linked intramolecularly with o-adenosine-5'-triphosphate and intermolecularly with o-adenosine, and conjugated with reduced glutathione (GSH). In this study, we compared the effects of our novel blood substitute and unmodified (U) Hb, by using allogenic plasma as the control, on human blood components: red blood cells (RBCs), platelets, monocytes (Mo), and low-density lipoproteins (LDLs). The pro-oxidant potential of both Hb solutions on RBCs was examined by the measurement of osmotic and mechanical fragility, conjugated dienes (CD), lipid hydroperoxides (LOOH), thiobarbituric acid reactants (TBAR-S), isoprostanes (8-iso PGF2alpha) and intracellular GSH. The oxidative modification of LDLs was assessed by CD, LOOH, and TBAR-S, and the degree of apolipoprotein (apo) B cross-linking. The effects of Hb on platelets have been studied by monitoring their responses to the aggregation agonists: collagen, ADP, epinephrine, and arachidonic acid. Monocytes were cultured with Hb solutions or plasma and tested for TNF-alpha and IL-1beta release, then examined by electron microscopy. Results indicate that native UHb initiates oxidative stress of many blood components and aggravates inflammatory responses of Mo. It also caused an increase in RBC osmotic and mechanical fragility (p < 0.001). While the level of GSH was slightly changed, the lipid peroxidation of RBC increased (p < 0.001). UHb was found to be a stimulator of 8-iso PGF2alpha synthesis, a potent modulator of LDLs, and an effective potentiator of agonist induced platelet aggregation. Contrarily, our novel blood substitute did not seem to induce oxidative stress nor to increase Mo inflammatory reactions. The osmotic and mechanical fragility of RBCs was similar to that of the control. Such modified Hb failed to alter LDLs, increase the production of 8-iso PGF2alpha, but markedly inhibited platelet aggregation. The effect of this novel blood substitute can be linked with the cytoprotective and anti-inflammatory properties of adenosine, which is used as a cross-linker and surface modifier, and a modification procedure that lowers the hemoglobin pro-oxidant potential.

Enhancing Nitrite Reductase Activity of Modified Hemoglobin: Bis-tetramers and Their PEGylated Derivatives

“…According to corporate-produced literature, glutathione reduces the potential oxidative stress inherent in HBOCs (41) while adenosine provides anti-inflammatory activity and contributes to vasorelaxation (42). It is not clear that these would be achieved in circulation since no evidence is yet available to support the claims.…”

Genetics, Biotechnology, and Developmental Biology

“…Previous studies have demonstrated that lipid radicals are able to inhibit anti-apoptotic genes which allow a cell to enter into apoptosis. In these experiments, the presence of oxidative stress was evaluated by measuring lipid hyperperoxides (LOOH) and reduced glutathione (GSH) levels according to established methods [21,22].…”

“…Established methods were utilized to measure oxidative stress conditions to determine the concentration of GSH [21]. In a normal cell, GSH is unable to cross the nuclear membrane, but during oxidative stress the oxidized form of GSH, oxidized glutathione (GSSG), is able to cross this structure.…”

“…GSSG is able to decrease the binding of p60/p65 complex of NF-κB to DNA, which reduces the pro-inflammatory cascade that is activated by NF-κB. The intracellular GSH levels were measured from cell homogenates [21]. The reaction of GSH with 5, 5' dithiobis and sulfhydryl compounds leads to a color change, producing a yellow pigment.…”

“…Nearly 30% of all buildings in developed nations are believed to have poor IAQ [17]. In addition to large occupancy buildings, 20-50% of modern homes are considered to have dampness and fungal contamination [19][20][21][22]. The Bureaus of National Affairs in 1992 identified that these conditions can lead to occupational concerns, as 30-70 million individuals are affected by SBS conditions.…”