Prolonging the shelf life ofLumbricus terrestriserythrocruorin for use as a novel blood substitute

Abstract

Limitations associated with the storage of red blood cells have motivated the development of novel blood substitutes that are able to withstand long-term storage at elevated temperatures. The hemoglobin of the earthworm Lumbricus terrestris (LtEc) is an attractive blood substitute candidate, since it is resistant to oxidation and aggregation during storage. Several factors were investigated to optimize the thermal and oxidative stability of LtEc during storage, including pH, antioxidant supplements, and deoxygenation. A strategy for the reduction of fully oxidized LtEc with antioxidants was also developed. Overall, LtEc was shown to have the highest thermal stability in Ringer's Modified Lactate solution with 10 mM HEPES at pH 7.0. Deoxygenation of the LtEc was also shown to significantly reduce oxidation of the ferrous heme iron (e.g., %Feafter 7 d at 37 °C = 75.7%). However, even in cases where oxidation does occur, the addition of 1.8 mM ascorbic acid (AA) was found to reduce 98.3% of the oxidized LtEc (37 μM heme). Most importantly, the oxygen transport properties of LtEc were unaffected by storage at high temperatures or oxidation followed by reduction with AA. These results show that LtEc can be stored at high temperatures (37 °C) without any significant loss of function.

Glutaraldehyde cross‐linking increases the stability of Lumbricus terrestris erythrocruorin

“…In contrast, the oxidation trends observed in Ringer's Modified Lactate Solution differed significantly from those observed in Tris buffer. Ringer's Lactate contains the antioxidants sodium lactate and N ‐acetyl l ‐cysteine, which decrease the oxidation rate of LtEc (but not HbA) . For example, HbA exhibited a much higher rate of oxidation (11.34 × 10 −3 h −1 ) in Ringer's Solution.…”

“…Ringer's Lactate contains the antioxidants sodium lactate and N-acetyl L-cysteine, which decrease the oxidation rate of LtEc (but not HbA). 30 For example, HbA exhibited a much higher rate of oxidation (11.34 3 10 23 h 21 ) in Ringer's Solution. In contrast, the oxidation rates of LtEc and all of the gLtEc samples significantly decreased by 2-3 orders of magnitude in Ringer's solution.…”

Therapeutic Potential of Hemoglobin Derived from the Marine Worm Arenicola marina (M101): A Literature Review of a Breakthrough Innovation

“…In the absence of protective enzymes such as superoxide dismutase (SOD) and catalase, Hb is oxidized to methemoglobin (MetHb), thus, compromising its ability to deliver O 2 [ 11 ]. Hb oxidation can compromise efficient O 2 binding to Hb through the formation of toxic heme degradation products, thereby, hampering optimal tissue perfusion [ 12 ]. Sufficient O 2 supply to tissues is crucial for optimal organ function, the complete failure of which can require an organ transplant.…”

“…Unfortunately, while many of these HBOCs showed promise in early clinical trials [ 15 , 28 , 30 , 31 ], they eventually failed Phase III clinical trials due to severe adverse effects, such as hypertension, myocardial infarction, stroke, renal damage and tissue toxicity, which are caused by rapid Hb oxidation and scavenging of the vasodilator nitric oxide by Hb [ 28 , 32 , 33 , 34 ]. These side effects can be attributed to the removal of intracellular mammalian Hb from the protective environment of the RBC and its release, even the modified forms, into the harsh extracellular environment of the bloodstream [ 12 ].…”

Initial biophysical characterization of Amynthas gracilis giant extracellular hemoglobin (HbAg)

“…There are some other interesting features related to Ec such as high thermal and oligomeric stability, oxidation resistance, cooperativity and affinity to bind oxygen, storage facility, and a large number of heme-containing polypeptide chains. All these characteristics make Ec particularly unique and interesting for studies aiming at biotechnological applications (Muzzelo et al 2018).…”