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A technologically improved variant of native stroma-free oxyhemoglobin (SFH) pretreated by carbontetrachloride and freeze-drying with 240 mM sucrose were reconstituted in a properly diluted ionic solution to reach the final concentration of 66 g oxyhemoglobin/L, osmolality 280-320 m0sm and pH 7.4. Cardioplegia of isolated rat heart was induced and maintained by this solution without recirculation for 3 h at 20 degrees C prior to heterotopic allo-transplantation of the graft. Evaluation of the survival and performance of each graft after 24 h and extent of tissue necroses indicated that the given standardly produced SFH variant ensured reproducible heart preservation from ischemic and reperfusion injury similarly as did the renown crystalloid cardioplegic solution CUSTODIOL.

Several approaches to the processing of native stroma-free hemoglobin solutions (SFH) were reconsidered regarding present requirements for SFH production and quality. Treatment of outdated red blood cells (RBCs) with chloroform and/or by pasteurization were evaluated for technical ease, speed and efficacy in removing stromata, phospholipids and non-heme proteins from RBC hemolysates. The influence of both procedures upon spontaneous hemiglobin formation in stored, preferably freeze-dried SFH was compared. Among other analytical methods, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS PAGE) and isoelectrofocusing were used for mutual comparison of the purification procedures. Pasteurized samples were significantly better purified, more homogeneous but also more susceptible to spontaneous oxidation, probably due to heat inactivation of enzymic scavengers of oxygen radicals. On the other hand, the chloroform-treated, unheated SFH samples were less purified from non-heme proteins, but were more stable. Fructose and sucrose were equally active in protecting SFH from oxidation during freeze-drying. At present, the easy chloroform treatment and freeze-drying of thus purified SFH with fructose of sucrose seems to offer a plausible technological compromise which merits further investigation.

The formation of heterogeneous fibers well observable under a magnifying glass in stirred "stroma-free" hemolysates of outdated banked erythrocytes was found to be predominantly due to random surface denaturation of both hemoglobin and non-heme proteins at the air-liquid interface. Electrophoretic analysis revealed that the precipitated and washed fibers contain residual membrane proteins as well as a certain amount of hemoglobin. The latter, however, largely remains in solution.

The reducing effect of ascorbic acid and of borohydride upon ferrihaemoglobin present in native and chemically modified human and bovine stroma-free hemoglobins was investigated. Ferrihaemoglobin which had been freshly prepared from oxyhaemoglobin by treatment with potassium ferricyanate was fully reduced to ferrohaemoglobin. Full reduction of ferrihaemoglobin, however, could not be achieved with those haemoglobin samples which had a partially deteriorated conformation due to long time storage or chemical modification.

A simple kinetic test with visual observation of hemoglobin solutions under 4 - 10x magnification was used to detect and roughly characterize a rapid formation of fine fibrous inhomogeneities in agitated "stroma-free" hemolyzates (SFH). In parallel SFH samples stored motionless for months, no such precipitate was observed. Hydrodynamic conditions are necessary to provoke a stepwise aggregation of small amounts of unstable filamentous nonhemoglobin molecules originating mostly from the stromata of erythrocytes and from constituents of other lysed blood cells. Numerous screening experiments mentioned here failed to remove significantly the "fiber-forming" substances from SFH or to prevent their precipitation. Development of a hydrodynamically stable and better purified SFH seems to be a prerequisite for further progress in the field of infusable SFH and its chemically modified variants (MSFH).

Sucrose is one of the most effective substances which protect haemoglobin from spontaneous methaemoglobin formation during lyophilization and subsequent storage. The dry haemoglobin-sucrose system was treated under different conditions of temperature (up to 85 degrees C), time of storage (up to 6 years) and residual moisture (less than 0.1-7.5% H2O), in order to reveal the main features of the hitherto unclear mechanisms of both spontaneous methaemoglobin formation and of the stabilizing effect of sucrose. In conclusion, a dry sucrose network was recognized as a significant support to the native ferrous structure of oxyhaemoglobin, while the presence of water molecules, of assumed peroxidic radicals and the action of thermal vibrations favour the oxidation and denaturation of haemoglobin.

The stability of experimental haemoglobin solution for infusion (HSI) was investigated during one year storage at temperatures 20 degrees to 25 degrees C, 4 degrees C, -8 degrees to -12 degrees C, and -195.8 degrees C, respectively. Main attention was devoted to the formation of precipitate, oxidation to haemiglobin and general changes in the absorption spectrum between 550 and 650 nm. Changes were almost negligible in HSI samples stored in the frozen state. This procedure now seems the most feasible to keep the HSI injectable for at least one year. Storage at 4 degrees C led to a marked increase of haemiglobin and to changes of the absorption spectrum. Storage at 20 degrees to 25 degrees C caused profound changes of all parameters tested. Addition of EDTA, ascorbic acid or glutathione, or storage in N2 atmosphere under the given conditions failed to increase the stability of HSI. Storage of HSI samples at pH values between 7.0 and 7.8 led to similar results.

The addition of stroma-free hemoglobin solution to a standard St. Thomas Hospital cardioplegic solution significantly protected the heart from ischemic damage compared to the effect of the same solution without added hemoglobin. An experimental model of rat heart cardioplegia and transplantation comprising heart arrest for three hours at 20 degrees C was used. The number of hearts performing strong contractions after cardioplegia with iso-oncotic oxyhemoglobin prior to transplantation was close to the results with histidine-buffered cardioplegic solution according to Bretschneider. Comparative biochemical model experiments in vitro confirmed that the positive effect of oxyhemoglobin was due predominantly to its buffering capacity. The role of oxygen transport to tissues by hemoglobin was limited only to the first minutes of cardioplegia since neither recirculation nor reoxygenation took place in the present experimental setting.