Evaluation of methemoglobin formation during the storage of various hemoglobin solutions.

Many researchers are trying to develop a blood substitute based on chemically modified human hemoglobin. In the process of making such solutions, we were faced with the problem of determining the best storage conditions to minimize oxidation of the solutions between the time of manufacture and use. Samples of stroma-free hemoglobin, purified A0 hemoglobin, and various cross-linked hemoglobins were stored for 8-12 months at +4 degrees C -20 degrees C, and -80 degrees C and were analyzed periodically for formation of methemoglobin (MetHb). Various suspending solutions were evaluated for their effects on the rate of MetHb formation, and the approximate rates of MetHb production per month were calculated. Short-term storage of hemoglobin solutions (< 14 days) can be done at +4 degrees C, but extended storage should be done at -80 degrees C with quick thawing. Salts minimize the hemoglobin oxidation during the stress of freeze-thaw operations. Storage at -20 degrees C. presents further problems and should be avoided.

High-performance liquid chromatographic evaluation of pyridoxyl 5'-phosphate hemoglobin derivatives produced by different reduction procedures.

Pyridoxylated hemoglobin derivatives have been studied by many investigators. In this study hemoglobin A0 rather than stroma-free hemoglobin was used as a starting material in order to reduce the number of proteins to A0 and A1c. Derivatives were characterized using a Synchropak Q300 strong anion-exchange column, a PolyCAT A weak cation-exchange column and a VYDAC reversed-phase high-performance liquid chromatographic column. Resulting peak profiles of these two ion-exchange separations demonstrated enhanced resolution and showed the presence of pyridoxylated hemoglobin products not previously described. We compared products from the reduction of these Schiff base derivatives using either sodium borohydride or sodium cyanoborohydride reduction procedures. The sodium cyanoborohydride reduction method produced a lower percentage of products with multiple-site pyridoxylation modifications than the sodium borohydride reduction process.

Reexamination of the polymerization of pyridoxylated hemoglobin with glutaraldehyde.

Pyridoxylated adult human hemoglobin (HbAo) was prepared using a one molar equivalent of pyridoxal 5-phosphate (PLP) per heme and reduced with either NaCNBH3 or NaBH4. A separate sample was pyridoxylated and passed through a mixed-bed ion exchange column without reduction. All three preparations had a P50 of 29 +/- 2 torr and a cooperativity of n = 2.4 +/- 0.1. These preparations, in both the oxy and deoxy forms, were then treated with 7 equivalents of glutaraldehyde per tetramer at pH 6.8 at 4 degrees C and at room temperature. The polymerization invariably reduced the P50 to 18 +/- 2 torr with Hill coefficients of less than 2. These solutions, with or without further reduction using NaCNBH3, all retained the PLP in differing amounts (2-3 moles/tetramer). Methemoglobin concentrations were increased during the polymerization reaction. The normal pyridoxylation procedure, using sodium borohydride reduction, resulted in a number of different molecular species. Polymerization with glutaraldehyde caused a further proliferation of molecular species that could not be separated by anion exchange chromatography or by isoelectric focusing. The extent of polymerization, estimated by gel exclusion chromatography and SDS polyacrylamide gel electrophoresis, was from 40 to 50%. Analysis of the reverse phase chromatograms, which separate the heme and the alpha- and beta-chains, showed extensive polymerization and distribution of the radioactively labeled PLP on the protein for all preparations. All of the polymerized and pyridoxylated samples were unstable, and showed different chromatographic patterns after storage at 4 degrees C for 1 month. Attempts to stabilize these preparations by further reduction with NaCNBH3 gave products with a lower P50 and lower cooperativity. When the reactions were conducted with a purified HbAo, heterogeneity was somewhat decreased compared to the normally used stroma-free hemoglobin, but a large number of molecular species were still formed.

A critical examination of the reaction of pyridoxal 5-phosphate with human hemoglobin Ao.

Pyridoxylated normal adult human hemoglobin (HbAo) has been prepared using both oxygenated and deoxygenated HbAo at pH 6.8 and room temperature without the addition of Tris to produce a mixture with P50 of 30 +/- 2 torr and a Hill coefficient of 2.3 +/- 0.1 similar to that of the isolated adult human hemoglobin from the red blood cell. Reduction of the pyridoxylated HbAo in the oxygen-ligated form by sodium borohydride gives unacceptable levels of methemoglobin (i.e., greater than 10%). Excessive foaming and methemoglobin formation can be partially avoided using deoxyHbAo. Reduction with sodium cyanoborohydride is much gentler and gives solutions with less than 5% methemoglobin. Both reducing agents give products with multiple components as shown by analytical chromatography. Radioautography on the isoelectric focusing gels of HbAo treated with 14C pyridoxal 5-phosphate (PLP) shows three major bands for the cyanoborohydride-reduced derivatives and a much more complex mixture of labeled molecules after the sodium borohydride reduction. When pyridoxylated hemoglobin is prepared without reduction, the preparation, after passage through a mixed-bed resin, contains 0.4 equivalents of PLP per heme, and has a P50 of 30 +/- 2 torr and an n value of 2.3 similar to the values found after reduction. Upon anion exchange resin chromatography, the PLP is removed, indicating that the reaction forms a reversible Schiff base. On standing at 4 degrees C for one month, this preparation produces a mixture of HbAo and pyridoxylated HbAo with the original P50. Methemoglobin increased to 3% during this incubation. After four months in the cold, the yield of a single chromatographic species is 70% with 20% methemoglobin. This fraction appears to be stable and can be passed through an anion exchange column without release of the PLP. Separation of the individual chains by reverse-phase chromatography indicates that the addition of PLP to HbAo is directed solely to the beta-chains. This is also the case for the cyanoborohydride reduced derivatives. When NaBH4 is used for the reduction, radioactively labeled PLP is found on both the alpha- and beta-chains.

High-performance liquid chromatographic analysis of ascorbate-2-phosphate, adenine and hypoxanthine in stored human blood.

A high-performance liquid chromatographic procedure was developed to assay for adenine, hypoxanthine, and ascorbate-2-phosphate in both red blood cells and plasma. The samples were diluted and filtered through Centriflo filter cones to remove most of the blood proteins before injection onto the column. The application of the technique to an experimental blood preservation study is illustrated.