Lowering of elevated tissue PCO2 in a hemorrhagic shock rat model after reinfusion of a novel nanobiotechnological polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase that is an oxygen and a carbon dioxide carrier with enhanced antioxidant properties

Abstract

Even though erythrocytes transport both oxygen and carbon dioxide, research on blood substitutes has concentrated on the transport of oxygen and its vasoactivity and oxidative effects. Recent study in a hemorrhagic shock animal model shows that the degree of tissue PCO(2) elevation is directly related to mortality rates. We therefore prepared a novel nanobiotechnological carrier for both O(2) and CO(2) with enhanced antioxidant properties. This is based on the use of glutaraldehyde to crosslink stroma free hemoglobin (SFHb), superoxide dismutase (SOD), catalase (CAT) and carbonic anhydrase (CA) to form a soluble PolySFHb-SOD-CAT-CA. It was compared to blood and different resuscitation fluids on the ability to lower elevated tissue PCO(2) in a 2/3 blood volume loss rat hemorrhagic shock model. Sixty minutes of sustained hemorrhagic shock at 30 mm Hg resulted in the increase of tissue PCO(2) to 95 mm ± 3 mmHg from the control level of 55 mm Hg. Reinfusion of whole blood (Hb 15 g/dL with its RBC enzymes) lowered the tissue PCO2 to 72 ± 4.5 mmHg 60 minutes after reinfusion. PolySFHb-SOD-CAT-CA (SFHb 10 g/dL plus additional enzymes) was more effective than whole blood in lowering PCO(2) lowering this to 66.2 ± 3.5 mmHg. Ringer's Lactated solution or polyhemoglobin lowered the elevated PCO2 only slightly to 87 ± 4.5 mmHg and 84.8 ± 1.5 mmHg, respectively. Moreover, ST-elevation for whole blood (Hb 15 g/dL) and PolySFHb-SOD-CAT-CA (Hb 10 g/dL) was respectively 12.8% ± 4% and 13.0% ± 2% of the control 60 minutes after reinfusion. Both are significantly better than those in the Ringer's lactated group and the PolyHb group. In conclusion, this novel approach for blood substitute design has resulted in a novel nanobiotechnological carrier for both O(2) and CO(2) with enhanced antioxidant properties.

Figure 1

Upper: Free molecules of hemoglobin, superoxide dismutase, catalase and carbonic anhydrase in solution. Lower: Glutaraldehyde is used to crosslink the free molecules in to a soluble nanobiotechnological complex of Polyhemoglobin-Superoxide dismutase-catalase-carbonic anhydrase.

Figure 2

Tissue PCO₂ in hemorrhagic shock (rats). Three time periods: (1). Hemorrhagic shock by removing 67% of total blood volume and maintaining the MAP at 30 mm Hg for 1 hour. (2) Reinfusion of one of the resuscitation fluids. (3) Followed for 1 hour after reinfusion. Resuscitation fluids are (A) Shed blood; (B) Ringer’s lactated solution 3 volumes of shed blood; (C) Polyhemoglobin (PolyHb) prepared with pure Hb containing no RBC enzymes and given at the concentration of Hb 10 g/dL, same volume as shed blood; (D) PolySFHb-SOD-CAT-CA prepared from RBC content (SFHb) enriched with additional superoxide dismutase (SOD), catalase (CA) and carbonic anhydrase (CA) given at the concentration of Hb10 g/dL in the same volume as shed blood.

Figure 3

Tissue PCO2 in hemorrhagic shock (rats). As above but with the following resuscitation fluids given in the same volume as shed blood. (A) PolyHb with no RBC enzymes as above but at a lower concentration of Hb 5 g/dL; (B) PolySFHb prepared from RBC content (SFHb) with RBC enzymes at the concentration of Hb 5 g/dL; (C) PolySFHb prepared with SFHb as B above but at a higher concentration of Hb10 g/dL; (D) PolySFHb-SOD-CAT-CA prepared with SFHb but with further enrichment of additional SOD, CAT and CA given at the concentration of Hb 5 g/dL.

Figure 4

This figure compares the effects on tissue PCO₂ of all the groups. As shown in graph: Ringer’s Lactated solution, PolyHb (10 g/dL), PolySFHb 10 g/dL, Blood 15 g/dL and PolySFHb-SOD-CAT-CA 10 g/dL). Those solutions with hemoglobin concentration of 5 g/dL are: 2 (PolyHb), 4(PolySfHb), 6(PolySFHb-SOD-CAT-CA).

Figure 5

MAP in hemorrhagic shock (rats). Three time periods: (1). Hemorrhagic shock by removing 67% of total blood volume and maintaining the MAP at 30 mm Hg for 1 hour. (2) Reinfusion of one of the resuscitation fluids. (3) Followed for 1 hour after reinfusion. Resuscitation fluids are (A) Shed blood; (B) Ringer’s lactated solution 3 volumes of shed blood; (C) Polyhemoglobin (PolyHb) prepared with pure Hb containing no RBC enzymes and given at the concentration of Hb 10 g/dL, same volume as shed blood; (D) PolySFHb-SOD-CAT-CA prepared from RBC content (SFHb) enriched with additional superoxide dismutase (SOD), catalase (CA) and carbonic anhydrase (CA) given at the concentration of Hb 10 g/dL in the same volume as shed blood.

Figure 6

MAP in hemorrhagic shock (rats). As in Figure 5 but with the following resuscitation fluids given in the same volume as shed blood.(A) PolyHb with no RBC enzymes as above but at a lower concentration of Hb 5 g/dL; (B) PolySFHb prepared from RBC content (SFHb) with RBC enzymes at the concentration of Hb 5 g/dL; (C) PolySFHb prepared with SFHb as B above but at a higher concentration of Hb10 g/dL; (D) PolySFHb-SOD-CAT-CA prepared with SFHb but with further enrichment of additional SOD, CAT and CA given at the concentration of Hb 5 g/dL.

Figure 7

This is a combined figure to compare the effects on MAP of all the groups. As shown in graph: Ringer’s Lactated solution, PolyHb (10 g/dL), PolySFHb 10 g/dL, Blood 15 g/dL and PolySFHb-SOD-CAT-CA 10 g/dL). Those solutions with hemoglobin concentration of 5 g/dL are: 2 (PolyHb), 4(PolySfHb), 6(PolySFHb-SOD-CAT-CA).

Figure 8

% of ST elevation 60 minutes after reinfusion compare to value before hemorrhagic shock : whole blood (Hb 15 g/dL); PolyHb (Hb 10 g/dL); PolySFHb (Hb 10 g/dL) and PolySFHb-SOD-CAT-CA (Hb 10 g/dL).