Oxygen delivery during extreme anemia with ultra-pure earthworm hemoglobin

Abstract

Aim: Lumbricus terrestris (earthworm) erythrocruorin (LtEc) is a naturally occurring extracellular hemoglobin (Hb) with high molecular weight (3.6MDa), low autoxidation rate, and limited nitric oxide (NO) dioxygenation activity. These properties make LtEc a potential candidate for use as red blood cell (RBC) substitute, i.e. Hb-based oxygen carrier (HBOC). Previous studies have shown that small amounts of LtEc can be safely transfused into mice, rats, and hamsters without eliciting major side effects. Therefore, this study was designed to understand oxygen (O(2)) transport to tissues and systemic/microvascular hemodynamics induced by LtEc during anemic conditions.

Main methods: Hamsters fitted with dorsal window chambers were hemodiluted to 18% hematocrit (Hct) using 6g/dL dextran 70kDa (Dex70). Hemodilution was then continued to 11% Hct using 10g/dL LtEc, 6g/dL Dex70 or 10g/dL human serum albumin (HSA). Blood pressure, heart rate, blood gas parameters, microvascular hemodynamics, microvascular blood flow, functional capillary density (FCD), intravascular pO(2) and perivascular pO(2) were studied.

Key findings: LtEc maintained blood pressure without inducing vasoconstriction while increasing microvascular perfusion and FCD relative to Dex70 and HSA. LtEc increased blood O(2) carrying capacity and maintained systemic and microvascular parameters without decreasing arteriolar diameter or increasing vascular resistance with during extreme anemia. LtEc increased O(2) delivery compared to conventional plasma expanders.

Significance: LtEc or synthetic molecules that replicate the characteristics of LtEc could be effective O(2) carriers with potential to be used in transfusion medicine to prevent tissue anoxia resulting from severe anemia.

FIGURE 1

Mean arterial pressure (MAP) and heart rate (HR) after extreme hemodilution with LtEc, HSA and Dex70. The broken line represents the baseline level. †, P < 0.05 relative to baseline; ‡, P<0.05 compared to Dex70; §, P<0.05 compared to HSA.

FIGURE 2

Relative changes to baseline in arteriolar and venular hemodynamics after extreme hemodilution with LtEc, HSA and Dex70. The broken line represents the baseline level. †, P < 0.05 relative to baseline; ‡, P<0.05 compared to Dex70; §, P<0.05 compared to HSA. Diameters (μm, mean ± SD) at baseline in each animal group were as follows: LtEc (arterioles (A): 62.1 ± 7.2, n = 48; venules (V): 62.6 ± 5.8, n = 46); HSA (A: 61.9 ± 9.1, n = 42; V: 63.6 ± 7.2, n = 46); and Dex70 (A: 62.7 ± 8.6, n = 48; V: 64.8 ± 7.4, n = 46). n = number of small blood vessels studied. RBC velocities (mm/s, mean ± SD) at baseline in each animal group were as follows (data was not presented in the figure, but used in the calculation of blood flow): LtEc (A: 4.3 ± 0.9; V: 1.9 ± 0.7); HSA (A: 4.3 ± 1.0; V: 2.0 ± 0.8); Dex70 (A: 4.4 ± 0.8; V: 2.1 ± 0.7). Blood flow (nL/s, mean ± SD) at baseline in each animal group were as follows: LtEc (A: 12.7 ± 5.3; V: 5.4 ± 2.1); HSA (A: 12.4 ± 4.2; V: 5.7 ± 2.1); Dex70 (A: 12.0 ± 4.7; V: 6.2 ± 2.4).

FIGURE 3

Functional capillary density (FCD) after extreme hemodilution with LtEc, HSA and Dex70. †, P < 0.05 relative to baseline; ‡, P<0.05 compared to 18% Hct. FCD (capillaries per unit of area, cm⁻¹) at baseline for LtEc were 112 ± 11, Dex70 were 114 ± 12 and for HSA were 109 ± 10, respectively.

FIGURE 4

Intravascular and perivascular partial pressure of oxygen after moderate hemodilution and extreme hemodilution with LtEc, HSA and Dex70. Intravascular and perivascular PO₂s for the tissues comprising the hamster window model are 51.8 mm Hg for arterioles, 32.7 mm Hg for venules and 21.7 mm Hg for perivascular areas.

FIGURE 5

Arterial oxygen delivery and extraction after extreme hemodilution with LtEc, HSA and Dex70. Oxygen transport is not directly measurable; however, it can be calculated using the measured parameters. Hatched portion of bar indicated the amount of oxygen transported by the LtEc.

FIGURE 6

LtEc pharmacokinetics after a 40% exchange transfusion.