A novel hemoglobin-adenosine-glutathione based blood substitute: Evaluation of its effects on human blood ex vivo

Chemically modified hemoglobin (Hb) solutions are under current investigati on as potential red cell substitutes. Researchers at Texas Tech University have developed a novel free Hb based blood substitute product. This blood s ubstitute is composed of purified bovine Hb cross-linked intramolecularly w ith o-adenosine-5'-triphosphte and intermolecularly with o-adenosine, and c onjugated with reduced glutathione (GSH). In this study, we compared the ef fects of our novel blood substitute and unmodified (U) Hb, by using allogen ic plasma as the control, on human blood components: red blood cells (RBCs) , platelets, monocytes (Mo), and low-density lipoproteins (LDLs). The pro-o xidant potential of both Hb solutions on RBCs was examined by the measureme nt of osmotic and mechanical fragility, conjugated dienes (CD), lipid hydro peroxides (LOOH), thiobarbituric acid reactants (TBAR-S), isoprostanes (8-i so PGF(2 alpha)) 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 moni toring their responses to the aggregation agonists: collagen, ADP, epinephr ine, and arachidonic acid. Monocytes were cultured with Hb solutions or pla sma and tested for TNF-alpha and Il-1 beta release, then examined by electr on microscopy. Results indicate that native UHb initiates oxidative stress of many blood components and aggravates inflammatory responses of Mo. It al so 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 PGF(2 alph a) synthesis, a potent modulator of LDLs, and an effective potentiator of a gonist induced platelet aggregation. Contrarily, our novel blood substitute did not seem to induce oxidative stress nor to increase Mo inflammatory re actions. The osmotic and mechanical fragility of RBCs was similar to that o f the control. Such modified Hb failed to alter LDLs, increase the producti on of 8-iso PCF2 alpha, but markedly inhibited platelet aggregation. The ef fect of this novel blood substitute can be linked with the cytoprotective a nd anti-inflammatory properties of adenosine, which is used as a crosslinke r and surface modifier, and a modification procedure that lowers the hemogl obin pro-oxidant potential.