Immobilized hemoglobin in the purification of hemoglobin-based oxygen carriers.

Chemically modified hemoglobins can be used as oxygen carriers in cell-free fluids provided that they have a low oxygen affinity and are stable towards dissociation into subunits. The latter species are undesirable because they are filtered rapidly through the kidneys, have renal toxicity and are characterized by a high oxygen affinity. A most important step in the preparation of hemoglobin-based oxygen carriers is therefore their purification from any dissociable material. Hemoglobin immobilized as alpha beta dimers on Sepharose lends itself naturally to this purpose as it is able to interact in a specific and reversible way with soluble alpha beta dimers. Hemoglobin affinity columns are very effective in the purification of cross-linked and pseudo-cross-linked human and bovine hemoglobin. The applicability of the technique is enhanced by the ease with which alpha beta dimers from different species cross-interact to yield hybrid alpha 2 beta 2 tetramers. It is shown that hemoglobin affinity columns may provide analytical information on the cross-linking reaction itself.

Intravascular retention and renal handling of purified natural and intramolecularly cross-linked hemoglobins.

Plasma half time of unmodified hemoglobin (UHb) and two intramolecularly cross-linked hemoglobins (alpha alpha XL and beta beta XL) was measured in anesthetized rats after an intravenous bolus of 20 mg.100 gm.-1 To rule out the possibility that differences among plasma half times might be caused by differences in acute effects on renal excretory function, glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were measured simultaneously with plasma half time. Experiments were also done to determine whether higher doses (60 to 100 mg-1.100 gm-1) of these compounds had a delayed effect (48 hours) on GFR or ERPF. Massive urinary excretion of UHb occurred; however, only 1% of the alpha alpha XL and none of the beta beta XL was excreted. Plasma half time of alpha alpha XL and beta beta XL averaged 3.3 hours, or four times longer than UHb. In no case did a decrease in GFR or ERPF occur. Instead, a transient increase in GFR, ERPF, urine flow, and systemic blood pressure was seen. Similar increases occurred after albumin administration, suggesting expansion of vascular volume as the initiating factor. Renal functions at 48 hours after 60 to 100 mg.100 gm-1 of UHb, alpha alpha XL or beta beta XL were not different from control (albumin). Intratubular hemoglobin casts or intravascular precipitates were not evident in acute or 48-hour studies. At 48 hours Perls' staining material was found in one alpha alpha XL specimen at 3 hours after administration. Perls' staining material was present in renal tubule cells in all but the albumin-treated kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of fructose and mannitol on dog renal cortical adenine nucleotide content, in vivo.

The renal cortex of anesthetized dogs was found to contain per gram lyophilized dry weight 9.5+/-0.6 micronmol ATP, 3.2+/-0.5 MICRONMOL ADP, 0.68+/-0.3 MICRONMOL AMP, and 22.5+/-4.4 micronmol Pi. These amounts were unchanged by the administration of moderate amounts of saline or hypertonic mannitol in load doses of about 20mmol/kg. Fructose in doses of 5 mmol/kg was found to cause a marked decrease in cortical ATP content. In doses of 20 mmol/kg fructose not only caused a decrease in ATP, but in total adenine nucleotides and Pi content as well. These results are consistent with the mode of action proposed for fructose, in rat kidney.