Diagnostic significance of urinary thiodiglycolic acid as a possible tool for studying the role of vitamins B12 and folates in the metabolism of thiolic substances.

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.

Improved preparation of soluble dry oxyhemoglobin purified by carbontetrachloride treatment of outdated packed erythrocytes.

A combination of treating outdated packed red blood cells (PRBCs) with carbontetrachloride, countercurrent dialysis and modified conditions of freeze-drying served to improve and standardize the technology of "stroma-free hemolysate" (SFH) in our laboratory. As a rule, proteins of the final product corresponded to 95% oxyhemoglobin, 3.5% methemoglobin and 1.5% non-hemoglobin ones. In freeze-dried samples stabilized with 0.24 molar saccharides, the weight proportions were 56% hemoglobin, 44% sucrose and/or 62% hemoglobin, and 38% fructose, respectively. If necessary, the saccharides could be rapidly removed from the easily reconstituted SFH by dialysis or chromatography. Analytical parameters were similar to those of chloroform-treated SFH stored dry at -12 degrees C for 4 months. However, the present procedure was easier and SFH samples remained unchanged after dry storage even at +4 degrees C for at least 13 months. This oxyhemoglobin product seems suitable for organ perfusion, further chemical modification and as an analytical standard.

Heart protection by cardioplegic solutions containing oxyhemoglobin pretreated by carbontetrachloride and freeze-drying with sucrose.

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.

Stroma-free hemoglobin solutions purified by chloroform and pasteurization.

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.

On the hydrodynamic instability of hemoglobin solutions.

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.

Hemoglobin solutions in experimental cardioplegia.

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.

Hydrodynamic instability of "stroma-free" hemoglobin.

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).

Trends in exploitation of packed red blood cells.

The following trends aim to a more efficient exploitation of packed red blood cells (PRBC): 1. Improvement of the operative distribution of PRBCs for transfusions before expiration. 2. Prolongation of the expiration time by monitoring the biochemical and physical processes during banking. Maintenance of native hemoglobin and restoration or substitution of substances involved in transport of energy and of oxygen are of utmost importance. Enzymic conversion of RBCs of blood group A, B to 0 is not supposed to leave laboratory scale soon. While cryo-conservation of RBCs with glycerine is known, freeze-drying of PRBCs remains a speculation. 3. Use of PRBCs after expiration as a raw material for products applicable in medicine and biochemistry. Stroma-free hemoglobin variants (SFH) are known as effective infusable oxygen carriers in experimental animal models. However, there is little convincing evidence on the metabolism and innocuity of SFH variants in human organism. Therefore, systemic infusion of SFH solutions is not yet acceptable to clinicians even in emergency situations. On the other hand, a broader use of SFH and its variants is anticipated and regarded as prospective in organ perfusion, cardioplegy and transplantation as well as in analytical biochemistry.

On the question of lowering the content of ferrihaemoglobin in infusable haemoglobin solutions.

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.

Haemoglobin solutions: reversibly bound oxygen and its effect upon the hypoxic heart.

Stroma-free solutions of human haemoglobin modified with pyridoxal-5-phosphate, glutaraldehyde, borohydride and serum albumin were injected into the artery of an isolated rat heart perfused with Krebs-Henseleit solution under hypoxic conditions. About 70% of the oxygen transported by the modified haemoglobin was found to be utilized for a marked increase in the force of heart contraction. The results were in general correlation to the analysis of oxygenation curves of haemoglobin samples under study and confirmed the oxygen offloading ability.