NMR studies of erythrocytes immobilized in agarose and alginate gels

Abstract

³¹P and ¹³C NMR were used to study the energy metabolism in perfused, human erythrocytes. The erythrocytes were immobilized in agarose threads, Ca‐ or Ba‐alginate beads, and Ba‐alginate‐coated agarose threads. Erythrocytes were easily washed out from the agarose threads, but not from alginate‐containing gels. Various small molecules, such as hypophosphite, dimethyl methylphosphonate, and methylphosphonate, were taken up from the perfusion medium in a normal manner. In addition, the 2,3‐bisphosphoglycerate (2,3‐DPG) chemical shifts were sensitive to the oxygen partial pressure suggesting that O₂ molecules were diffusing through the gel and modifying the binding of 2,3‐DPG to hemoglobin. A combination of inosine and pyruvate stimulated the synthesis of 2,3‐DPG. but only if inorganic phosphate was present in the perfusion medium. Inosine only resulted in a dramatic rise in the intracellular sugarphosphate concentrations. Furthermore, [2‐¹³C]glucose was converted to [2‐¹³C]lactate by immobilized cells at a rate which was comparable to that in a control suspension. In summary, immobilization in Ba‐alginate‐coated agarose threads was an efficient way of trapping human erythrocytes for whole cell NMR investigations. © 1992 Academic Press, Inc.