X-Message-Number: 15406
Date: Sun, 21 Jan 2001 10:39:01 -0800 (PST)
From: Doug Skrecky <>
Subject: reversible chemical fixation?

Title
  Reversible cross-linking and CO treatment as an approach in red cell
  stabilization.
Source
  Cryobiology.  40(4):343-59, 2000 Jun.
Abstract
  We explored the use of the reversible cross-linking reagent dimethyl
  3,3-dithiobispropionimidate (DTBP) in combination with CO treatment as an
  approach to stabilizing erythrocyte structure and function. Erythrocytes were
  cross-linked with different concentrations of DTBP for different times. DTBP
  increased erythrocyte osmotic stability, blocked lysolecithin-induced
  echinocytosis, and decreased erythrocyte deformability in a concentration-
  and time-dependent manner. Reversal of the cross-linking with the reducing
  agent dithioerythritol (DTE) restored osmotic fragility and response to
  lysolecithin as well as deformability. Complete reversal, however, is a
  function of the DTBP concentration and the time of cross-linking. The effects
  of cross-linking with 5 mM DTBP for 1 h were completely reversible after
  treatment with 10 mM DTE for 20 min. Longer incubation times or higher
  concentrations of DTBP resulted in partial reversal by DTE of the effects
  produced by DTBP. Cross-linking and reversal only slightly reduced the ATP
  content. The hemoglobin contained in the cross-linked and reversed cells
  could still undergo reversible oxygenation and deoxygenation. Erythrocytes
  were pretreated with CO, cross-linked with 5 mM DTBP for 1 or 3 h, loaded
  with a solution containing 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 cross-linked with 5 mM
  DTBP for 3 h and freeze-dried to a final water content of
  10-15%. Non-cross-linked cells lysed 100% on rehydration in distilled water.
  No methemoglobin (MetHb) formation as a result of
  freeze-drying was detected in CO-treated
  cells. In non-CO-treated cells 20% of the Hb was converted to MetHb.
  Copyright 2000 Academic Press.

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