The long term goal is to extend the useful shelf life of human red blood cells.
The specific aims are: l) to study the effect of ammonium (NH4+) and phosphate (Pi) on nucleotide and carbohydrate metabolism of RBCs; 2) to study membrane vesiculation and aminophospholipid flip flop during blood bank storage; 3) to identify correlated changes in the phosphinositides; 4) to ascertain the development of protein oxidation during storage; 5) to perform autologous in vivo viability studies. Units of packed red blood cells (PRBCs) will be stored with experimental additive solutions (EASs) in which the concentrations of NH4+ and Pi will be varied. The in vitro studies will include measurements at intervals up to 12 weeks of adenosine triphosphate 2,3-diphosphoglycerate, mean corpuscular volume, hemolysis, vesiculation and pH. 51Cr-labeled RBCs will be used in paired viability studies comparing selected EASs with a commercial additive solution, Adsol. For the metabolic studies, adenine- 8-14c will be used to measure the turnover of purine nucleotides and nucleosides. The levels of glucose-6-PO4 and fructose-6-PO4 will be used as indicators of phosphofructokinase activity. The ratios of lactate to pyruvate and the oxidized to the reduced form of nicotinamide adenine dinucleotide (NAD:NADH) will be used to gauge the activity of 3- phosphoglyceraldehyde dehydrogenase. Trinitrobenzenesulfonic acid will be used to quantitate the change in accessibility of phosphatidyl serine and ethanolamine during storage. Thin layer chromatography will be performed to separate and quantify the phosphinositides. Spectrin oxidation will be assessed using thiol disulfide exchange chromatography followed by gel electrophoresis. These studies will serve to enhance understanding of the metabolic changes affecting RBCs during blood bank storage. The resulting health benefits will be: 1) more effective operation of autologous donor programs. Longer effective shelf life of RBCs will make it possible to store more units preoperatively if needed and permit longer intervals between donations when the patient's clinical status makes this desirable; 2) the longer shelf life will make it possible to maintain the blood inventories of outlying hospitals more effectively with less outdating. Loss of blood by outdating still averages 10 percent in such situations.
