Reversible cross-linking and CO treatment as an approach in red cell stabilization

We explored the use of the reversible cross-linking reagent dimethyl 3,3-di thiobispropionimidate (DTBP) in combination with CO treatment as an approac h to stabilizing erythrocyte structure and function. Erythrocytes were cros s-linked with different concentrations of DTBP for different times. DTBP in creased erythrocyte osmotic stability, blocked lysolecithin-induced echinoc ytosis, and decreased erythrocyte deformability in a concentration- and tim e-dependent manner. Reversal of the cross-linking with the reducing agent d ithioerythritol (DTE) restored osmotic fragility and response to lysolecith in as well as deformability. Complete reversal, however, is a function of t he DTBP concentration and the time of cross-linking. The effects of cross-l inking with 5 mM DTBP for 1 h were completely reversible after treatment wi th 10 mM DTE for 20 min. Longer incubation times or higher concentrations o f DTBP resulted in partial reversal by DTE of the effects produced by DTBP. Cross-linking and reversal only slightly reduced the ATP content. The hemo globin contained in the cross-linked and reversed cells could still undergo reversible oxygenation and deoxygenation. Erythrocytes were pretreated wit h CO, cross-linked with 5 mM DTBP for 1 or 3 h, loaded with a solution cont aining 500 mM glucose for 24 h, and freeze-dried in a medium containing 15% (w/v) albumin. Rehydration followed in distilled water. Complete recovery, measured as the percentage of free hemoglobin, was achieved for cells cros s-linked with 5 mM DTBP for 3 h and freeze-dried to a final water content o f 10-15%, Non-cross-linked cells lysed 100% on rehydration in distilled wat er. No methemoglobin (MetHb) formation as a result of freeze-drying was det ected in GO-treated cells. In non-GO-treated cells 20% of the Hb was conver ted to MetHb. (C) 2000 Academic Press.