Hemoglobin (Hb) has long been proposed for use as a blood substitute. In order to overcome its disadvantages of massive renal excretion and short plasma half-life, Hb was conjugated to dextran yielding a dextran-hemoglobin conjugate (Dx-Hb). Methods to stabilize and right-shift the conjugates had been invented, and exchange transfusions with Dx-Hb in dogs and macaques performed. However, hitherto the flow properties of the conjugate have not been investigated. In this study, Dx-Hb with varying viscosities (1.98 - 2.60 cps) and effects on erythrocyte sedimentation rate (ESR) (12 - 53 mm/hr) were synthesized, and the precise relationship between ESR enhancement and the molecular size of modified Hb was delineated. It was found that red blood cell aggregation was induced by overly large gl...[ Read more ]

Hemoglobin (Hb) has long been proposed for use as a blood substitute. In order to overcome its disadvantages of massive renal excretion and short plasma half-life, Hb was conjugated to dextran yielding a dextran-hemoglobin conjugate (Dx-Hb). Methods to stabilize and right-shift the conjugates had been invented, and exchange transfusions with Dx-Hb in dogs and macaques performed. However, hitherto the flow properties of the conjugate have not been investigated. In this study, Dx-Hb with varying viscosities (1.98 - 2.60 cps) and effects on erythrocyte sedimentation rate (ESR) (12 - 53 mm/hr) were synthesized, and the precise relationship between ESR enhancement and the molecular size of modified Hb was delineated. It was found that red blood cell aggregation was induced by overly large glutaraldehyde-polymerized hemoglobin (Poly-Hb) or Dx-Hb molecules. On the other extreme, excessively small molecules would result in high renal excretion levels. Employing the two indices, namely ESR enhancement and renal excretion level, a molecular weight window was defined for each of Poly-Hb and Dx-Hb, within which no disturbance of ESR or renal excretion was observed. It was determined that the effective molecular size for Dx-Hb or right-shifted dextran-hemoglobin (rsDx-Hb) for this purpose is 100 kD to 350 kD. When some elevation of ESR is desired, the effective size could be upwards from 350 kD.

In order to further define the clinical usefulness of the different sizes of conjugates, excretion by rabbits and exchange transfusion in dogs were performed. Dextran-hemoglobin synthesized from dextran of 10 kD (Dx(Tl0)-Hb) showed basically the same excretion rate as Dx-Hb synthesized from larger dextrans. The excretion of Dx-Hb was less than 8%, whereas for unmodified Hb it was about 40%. The hematocrit could be lowered to less than 2% with the refined rsDx-Hb, and the dogs survived essentially complete exchange transfusion. Both in vivo and in vitro methods were employed to confirm the hypothesis that Dx-Hb or rsDx-Hb could bind sufficient nitric oxide to increase vascular tone. This allows a deeper understanding of the intrinsic physiological properties of the conjugation product.

Researches in defining the manufacturing parameters such as the choice of starting size of dextran and the synthetic conditions for the conjugation were performed. With the use of Dx(Tl0) and diafiltration (the retentate of 50 kD cartridge) to select the desirable size of bromodextran, optimal preparation of Dx-Hb of varying size could be obtained. The production method is also cost-effective and amenable to large scale production.