Biophysical Properties and Oxygenation Potential of High-Molecular-Weight Glutaraldehyde-Polymerized Human Hemoglobins Maintained in the Tense and Relaxed Quaternary States

Abstract

Recent clinical evaluation of commercial glutaraldehyde-polymerized hemoglobins (PolyHbs) as transfusion solutions has demonstrated several adverse side effects. Chief among these is the hypertensive effect. Fortunately, previous studies have shown that the hypertensive effect can be attenuated by removing free hemoglobin (Hb) and low-molecular-weight (low-MW) PolyHbs from the PolyHb mixture. In this work, polymerized human Hb (PolyhHb) solutions were synthesized in two distinct quaternary states with high MW and subjected to extensive diafiltration to remove free Hb and low-MW PolyhHb components (<500 kDa). The resultant PolyhHb solutions possessed high MW, distinct quaternary state, distinct reactivities with O 2 and CO, similar NO deoxygenating rate constants, distinct autoxidation rate constants, high viscosity, and low colloid osmotic pressure. To preliminarily assess the ability of PolyhHb solutions to oxygenate surrounding tissues fed by a blood vessel, we evaluated the ability of PolyhHbs to transport O 2 to cultured hepatocytes in a mathematical model of a hollow fiber bioreactor. The structure of individual hollow fibers in the bioreactor is similar to that of a blood vessel and provides an easy way to assess the oxygenation potential of PolyhHbs without the need for expensive and time-consuming animal studies. It was observed that PolyhHbs with low O 2 affinities were more effective in oxygenating cultured hepatocytes inside the bioreactor than high O 2 affinity PolyhHbs. Taken together, our results show that it is possible to synthesize high-MW PolyhHbs with no free Hb and low-MW PolyhHb components that are capable of transporting O 2 to cultured cells/tissues.

Mixtures of tense and relaxed state polymerized human hemoglobin regulate oxygen affinity and tissue construct oxygenation

“…The current study expands upon the work of Zhang et al [ 17 ] and Zhou et al [ 18 ], who synthesized and characterized the biophysical properties of bovine and human PolyHbs in either T- or R-state. In this study, pure T- and R-state polymerized human Hb (PolyhHb) solutions were synthesized, characterized and mixed at different molar ratios to yield PolyhHb mixtures with varying O 2 affinities and biophysical properties.…”

“…Deoxygenated and oxygenated hHb were polymerized with glutaraldehyde using methods developed previously [ 16 , 17 ] to yield 35:1 (molar ratio of glutaraldehyde to hHb) tense (T) and 30:1 relaxed (R) state PolyhHb, respectively.…”

“…After 35–45 min of degassing, samples were drawn from the bottle using a long stainless steel needle to measure the pO 2 of the hHb solution using a Rapidlab 248 (Siemens, Malvern, PA) blood gas analyzer. When the measured pO 2 of the hHb solution dropped to < 20 mm Hg, Na 2 S 2 O 4 was added to the reaction vessel to remove residual O 2 from the hHb solution [ 17 ]. 300 mg Na 2 S 2 O 4 was dissolved in 300 mL N 2 sparged PBS (0.1 M, pH 7.4) at room temperature, the Na 2 S 2 O 4 solution was added to the hHb solution dropwise using a pump set to a flowrate of 0.1 mL/s.…”

Next‐generation polymerized human hemoglobins in hepatic bioreactor simulations

“…Human hemoglobin (hHb) was first purified via TFF as described by Palmer et al 23 Glutaraldehyde was used as the crosslinking agent at a 30:1 molar ratio of glutaraldehyde to hHb as described previously. 24,25 The hHb was diluted to 40 ± 5 g/L in phosphate-buffered saline (PBS) (0.1 M, pH 7.4) and filtered through a 0.2 μm polysulfone (PS) TFF module to remove any large aggregates formed during cryogenic storage.…”

Effects of Hemoglobin-Based Oxygen Carriers on Blood Coagulation

“…Also, at clinically relevant doses, HBOCs do not cause more platelet dysfunction compared to other resuscitative fluids. The extent of the vasoconstriction and hypertension decreases as the size of the HBOC increases [ 29 ]. There is some evidence that shows that p50 is better, although there is no definite proof, there is some evidence.…”