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.

We have found that the determination of thiodiglycolic acid (TDGA) in urine may help to characterize metabolic imbalance of substances participating in methionine synthesis, which leads to hyperhomocystinuria. From the metabolic scheme, based on a proper combination of known facts, we attempted to theoretically explain and to demonstrate the possibilities of TDGA formation via different ways of homocysteine transformation. This scheme was used in evaluating the results obtained by testing urine of a woman suffering from impaired function of methionine synthase reductase (CblE type of homocystinuria). The amount of TDGA excreted in her morning urine was very sensitive to the changes in her treatment based upon a combination of N5-formyl tetrahydrofolate, betaine and vitamin B12. Vitamin B12 given in the evening either alone or together with betaine increased the TDGA excretion in the morning urine up to ten times. On the other hand, in the absence of vitamin B12, betaine in combination with N5-formyl tetrahydrofolate hindered the appearance of TDGA in the morning urine. Generally, the determination of TDGA in urine of an appropriately pretreated patient may indicate the degree of success of the treatment.

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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.

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).