Studies on Avian Erythrocyte Metabolism. XIII. Changing Organic Phosphate Composition in Age-Dependent Density Populations of Chicken Erythrocytes

Abstract These studies were performed to determine whether the erythrocytes (RBC's) of chickens are able to alter the concentrations of organic phosphates, particularly inositol pentakis (dihydrogen phosphate ) (inositol-P5 ), in response to anemia. Acute anemia with 33.5% reticulocytes (retics) in the peripheral blood of 4-month-old White Leghorn cockerels was produced after two successive days of controlled bleeding. Five populations of RBC's were prepared from blood of these anemic birds based upon age-dependent density by angle rotor centrifugation. The relative increase in age of Fraction 1 (younger cells) through Fraction 5 (oldest cells) was verified by increased hemoglobin concentration and decreased retic count and cell volume. The acid aqueous-soluble phosphates from each RBC fraction were extracted, fractionated by ion exchange column chromatography, and the concentration of inorganic phosphate (Pi), adenosine triphosphate (ATP), and inositol-P5 quantitated by wet-ash phosphate analysis. Determination of Pi, ATP, and inositol-P5 in Fraction 1 (younger cells, 7.4% retics) from the initial (Day 1) blood withdrawn gave 4.3, 5.7, and 18.6 jumoles Pi/ml RBC, respectively. On Day 4 of controlled-bleeding, Pi, ATP, and inositol-P5 concentrations in Fraction 1 (95.1% retics) were 12.7, 10.6, and 12.3 μmoles Pi/ml RBC, respectively. On Day 11, after 1 week recovery from controlled-bleeding, the values for Pi, ATP, and inositol-P5 in Fraction 1 (37.7% retics) were 5.0, 5.4, and 19.0 μmoles Pi/ml RBC, respectively, which were similar to the values before bleeding. It appears that retics entering the circulation contain approximately 60% of the level of inositol-P5 in the mature RBC and that synthesis and final accumulation of inositol-P5 occurs during maturation of retics in circulation. No cell fraction had a concentration of inositol-P5 greater than that of normal mature RBC's. The concentration of 2,3-bisphosphoglycerate (2,3-P2-glycerate) was measured enzymatically on each cell fraction and no concentration greater than .2 μmoles/ml RBC was found. The molar ratio of inositol-P5 to hemoglobin in the five density fractions from blood on Day 1 of controlled-bleeding ranged between .81 and .88; the enriched retic population (Fraction 1, Day 4) had a ratio of .64. Our results indicate that inositol-P5 concentrations are not readily altered in response to acute anemia and that its regulation of hemoglobin function in birds is not as adaptable to changing needs of the organism as is the 2,3-P2-glycerate regulation of oxygen binding and delivery in the mammalian system.