Incorporation Of Inositol Hexaphosphate Research Articles

Low-O(2) affinity erythrocytes improve performance of ischemic myocardium.

O(2) transport and O(2) diffusion interact in providing O(2) to tissue, but the extent to which diffusion may be critical in the heart is unclear. If O(2) diffusion limits mitochondrial oxygenation, a change in blood O(2) affinity at constant total O(2) transport should alter cardiac O(2) consumption (VO(2)) and function. To test this hypothesis, we perfused isolated isovolumically working rabbit hearts with erythrocytes at physiological blood-gas values and P(50) (PO(2) required to half-saturate hemoglobin) values at pH of 7.4 of 17 +/- 1 Torr (2,3-bisphosphoglycerate depletion) and 33 +/- 5 Torr (inositol hexaphosphate incorporation). When perfused at 40 and 20% of normal coronary flow, mean VO(2) decreased from the control value by 37 and 46% (P < 0.001), and function, expressed as cardiac work, decreased by 38 and 52%, respectively (P < 0.001). Perfusion at higher P(50) during low-flow ischemia improved VO(2) by 20% (P < 0.001) and function by 36% (P < 0.02). There was also modest improvement at basal flow (P < 0.02 and P < 0.002, respectively). The improvement in VO(2) and function due to the P(50) increase demonstrates the importance of O(2) diffusion in this cardiac ischemia model.

Low‐oxygen‐affinity red cells produced in a large‐volume, continuous‐ flow electroporation system

Human red cells containing inositol hexaphosphate (IHP) have a lowered O2 affinity, though they are able to bind and carry about the same amount of oxygen as native cells. These modified cells therefore deliver oxygen more efficiently to the tissues, which is a property of potential clinical utility. Investigators set out to devise a system and procedure by which large volumes of IHP-containing red cells, suitable for transfusion, could be produced quickly and efficiently. The encapsulation of IHP into human red cells by use of several variations of static electroporation was performed to define the conditions necessary for optimal IHP incorporation and cell survival. These conditions were used as a starting point for optimization of a flow electroporation system. When fresh human red cells in a 35 mM IHP solution are subjected to three exponential pulses of field strength of 2.98 +/- 0.064 kV per cm per pulse and pulse length of 2.0 +/- 0.2 msec per pulse while flowing through a cooled electroporation chamber, the condition of the resultant cells, according to the criteria used here, is optimized. After storage for 24 hours in plasma at 37 degrees C, the cells show more than 85-percent survival (in vitro) and hematologic indices nearly identical to those of unpulsed control cells. The p50 value of these cells, however, has doubled to 50.4 +/- 2.0 torr. The processing time for 1 unit of blood is 90 minutes. These data indicate that the system described here can efficiently produce low-oxygen-affinity red cells in volumes that are useful in clinical applications.

Hemodynamic changes induced by low blood oxygen affinity in dogs

Increased tissue oxygen delivery may play a role in the increased vascular resistance that develops in volume-expanded hypertension. This hypothesis was tested by decreasing the affinity of hemoglobin for oxygen in dogs to increase unloading of oxygen to the tissues. Six chronically instrumented dogs were studied before and for 7 days after partial exchange transfusion with red blood cells modified by incorporation of inositol hexaphosphate, which, 1 h after exchange, increased the PO2 value at which hemoglobin is half-saturated with oxygen (P50) to 38.8 +/- 2.1 mmHg from a control value of 31 +/- 1.5 mmHg. Cardiac output (electromagnetic flowmeter) fell to 92.5 +/- 7.4 ml.kg-1.min-1 after 2-4 h from control values between 120.2 +/- 5.7 and 125.8 +/- 4.6 ml.kg-1.min-1. One day later, cardiac output was still significantly decreased to 104.0 +/- 5.9 ml.kg-1.min-1. As P50 returned to control over the next few days, so did cardiac output. Two to four hours after exchange, total peripheral resistance was increased to 1,144 +/- 73 mmHg.l-1.kg.min from control values between 762 +/- 26 and 790 +/- 32 mmHg.l-1.kg.min. It was still increased to 993 +/- 51 mmHg.l-1.kg.min after 1 day. Oxygen consumption did not change significantly. Cardiac output and peripheral resistance changes were significantly different from those measured in a control group of six dogs receiving exchange transfusion with sham-shifted red blood cells without significant P50 changes. These results suggest that an increase in tissue oxygen delivery can raise total peripheral resistance in dogs in the absence of primary changes in fluid volumes, blood flow, or blood pressure.

Preparation of low-affinity red cells with dimethylsulfoxide-mediated inositol hexaphosphate incorporation: hemoglobin and ATP recovery using a continuous-flow method.

Incorporation of IHP into red cells decreases oxygen affinity as a result of the binding of this compound to the 2,3-DPG site of hemoglobin. This investigation describes a continuous-flow method which utilizes the osmotic pulse technique to transport IHP into RBC. Using this procedure, it is possible to obtain a significant increase in P50 while maintaining in vitro cellular integrity. For example, IHP incorporation sufficient to cause an increase in the P50 of 20 mm Hg may be achieved with recovery of approximately 75% of the hemoglobin and with maintenance of ATP levels compatible with good viability. The continuous-flow method allows uniform treatment of large, unit-size volumes of red cells with a relatively small quantity of reagents. The final cell product is macrocytic/hypochromic with an increased number of stomatocytes.

Incorporation of inositol hexaphosphate into red blood cells mediated by dimethyl sulfoxide

Inositol hexaphosphate (IHP) binds to deoxyhemoglabin and markedly decreases the affinity of hemoglobin for oxygen. We introduce here a method for incorporating this polyphosphate into erythrocytes, thus preparing very low affinity cells for use in respiration research. The method uses dimethyl sulfoxide (DMSO) to facilitate entry of IHP. The cells are exposed to a high concentration of DMSO which is rapidly diluted with IHP solution. During this dilution the cells become leakly and IHP enters. The influence of several variables at each step of the process has been investigated and the data support a transient osmotic gradient mechanism for IHP incorporation.

Microencapsulation IV: Cross-Linked Hemoglobin Microcapsules

Hemoglobin microcapsules were prepared through cross-linking of hemoglobin itself with various acyldichlorides. Variations in the reticulation conditions were performed in order to ameliorate the oxygen dissociation curve, the mean diameter, and the possibility for the microcapsules to be lyophilized. With terephthaloylchloride, as the cross-linking agent, incorporation of inositol hexaphosphate and glucose, followed by stabilization through glutaraldehyde and using a high stirring speed, allowed preparation of stable hemoglobin microcapsules, 5 μm in diameter, which suffered rapid lysis by proteases. They were able to ensure oxygen transfer: the dissociation curve was sigmoidal with a p50 = 13 mm Hg. They retained these properties after lyophilization followed by rehydration.

Incorporation of inositol hexaphosphate into intact red blood cells

Fluid charged lipid vesicles loaded with an inositol hexaphosphate solution were used to transport this allosteric effector into human intact red blood cells. Rate and extent of uptake of the vesicles and of the effector by the red blood cells were measured as changes in the O2 half-saturation pressure and the 31P-NMR spectra of the intracellular inositol hexaphosphate-haemoglobin complex.