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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.

A combination of treating outdated packed red blood cells (PRBCs) with carbontetrachloride, countercurrent dialysis and modified conditions of freeze-drying served to improve and standardize the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.

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.

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.

Mouse cells were fractioned by adherence chromatography on glass beads. The fractionation of spleen cells yielded 5 different fractions, whose transplantation effect was tested by the method of Till and McCulloch. The results of these transplantation tests showed that the chosen form of adherence chromatography was not sufficiently successful, since none of the resultant fractions contained solely colony-forming units (CFU) and in none of them were CFU present in an exeptionally high concentration.

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 possibilities of separation of haemopoietic cells from lymphocytes capable of eliciting the graft-versus-host reaction through column chromatography were investigated. Strain-A mouse spleen cells were fractionated into the adherent and non-adherent fraction on Sephadex G-25, glass bead columns and glass beads coated with antibody against mouse globulin. Increased numbers of cells forming haemopoietic colonies were found in the cell fraction which did not adhere to the antibody-coated glass beads and in cells reversibly adhering to glass beads. No significant decrease in local graft-versus-host reaction was found in any fraction obtained, and the prolonged survival of irradiated semiallogeneic recipients was observed in both fractions obtained on Sephadex G-25 columns.

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 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.