Hypervolemic infusion of Lumbricus terrestris erythrocruorin purified by tangential‐flow filtration

Abstract

Background The hemoglobin of the Earthworm Lumbricus terrestris (also known as erythrocruorin, or LtEc) is a naturally occurring high molecular weight protein assembly (3.6 MDa) that is extremely stable, resistant to oxidation, and transports oxygen similarly to human whole blood. Therefore, LtEc may serve as an alternative to donated human red blood cells, however, a suitable purification process must be developed to produce highly pure LtEc on a large scale that can be evaluated in an animal model to determine the safety and efficacy of LtEc. Materials and Methods We used tangential flow filtration (TFF), an easily scalable and affordable technique, to produce highly pure LtEc from Earthworms. The purity, yield, methemoglobin level, viscosity, colloid osmotic pressure, O2 binding equilibria, and ligand binding kinetics of the purified LtEc was measured in vitro. The purified LtEc product was then evaluated in hamsters using a hypervolemic infusion model to establish its basic biocompatibility and detect any changes in microcirculatory and systemic parameters. Results TFF was able to produce LtEc with high purity and yield (5–10 grams per 1000 worms). The purified LtEc product did not elicit hypertension or vasoconstriction when infused into hamsters. Conclusion LtEc may be easily purified and safely transfused into hamsters in small amounts (0.5–1.5 g/dL final concentration in blood) without any noticeable side-effects. Therefore, LtEc may serve as a very promising oxygen carrier for use in transfusion medicine.

Polyethylene Glycol Camouflaged Earthworm Hemoglobin

“…The MALDI mass spectrum of LtEc and PEG-LtEc (Fig 1A) show similar to LtEc mass spectra existing in the literature [18]. For LtEc, monometers and linkers appear near their expected MW (16 kDa and 24–32 kDa), respectively.…”

“…[26] In addition, all the animals used in this study showed no allergic reactions to PEG-LtEc or LtEc, which confirms that ultra-purification of LtEc eliminates all harmful substances. [14, 15, 18]…”

“…PEG-LtEc did not cause vasoconstriction or hypertension compared to small polymerized Hbs solutions. [18] Instead, PEG-LtEc appeared to slightly relax small blood vessels. The high viscosity of PEG-LtEc (1.9 cP) relative to plasma viscosity (1.2 cP), may also help to produce vasodilation, as plasma viscosity increases endothelial wall shear stress on the blood vessel wall, which stimulates endothelial cell production of vasodilators, including NO and prostacyclin.…”

“…Samples were prepared for Matrix Assisted Laser Desorption Ionization (MALDI) by diluting LtEc and PEG-LtEc to 1 mg/mL, then mixing them in a 1:1:1 ratio with 1M HCl and a matrix solution of 50% v/v acetonitrile saturated with sinapic acid [18]. Cytochrome C (12,361 g/mol), apomyoglobin (16,952 g/mol), and human serum albumin (66,437 g/mol) were used as molecular weight (MW) standards on a Bruker FLEX MALDI-TOF (Bruker Daltonics, Billerica, MA) and analyzed in the linear mode [18].…”

“…Cytochrome C (12,361 g/mol), apomyoglobin (16,952 g/mol), and human serum albumin (66,437 g/mol) were used as molecular weight (MW) standards on a Bruker FLEX MALDI-TOF (Bruker Daltonics, Billerica, MA) and analyzed in the linear mode [18]. …”