Null-point titration measurements of free magnesium in stored erythrocytes

Free intracellular magnesium concentration (Mg/sub i/) was measured in stored human erythrocytes, using null-point titration with ionophore A23187. For cells stored <10 days in citrated preservation media (CPDA), Mg/sub i/ was 311 +/- 41 μM (mean +/- SD, N=7), whereas for cells stored 22-48 days it was 483 +/- 64 μM (N=12). This is in contrast to their earlier measurements by 31P NMR spectroscopy, which showed a decrease in Mg/sub i/ with storage. However, the NMR measurements are performed with no pretreatment of the cells, while the null-point method requires an initial washing step, which alters pH/sub i/ and may also alter Mg/sub i/. The titration-measured Mg/sub i/ values are still surprisingly low for long-stored cells, considering that depletion of ATP and 2,3-DPG should release bound Mg. Using the titration-measured Mg/sub i/ values along with measurements of total Mg, ATP, and 2,3-DPG, they estimate that an additional buffer contains about 47% of total Mg in cells stored <10 days, and 67% of total Mg in cells stored >21 days. Mg/sub i/ determinations by both 31P NMR and null-point titration thus indicate that erythrocyte Mg is largely bound to a high-capacity, low-affinity buffer whose relative importance increases during cell storage. Discrepancies between the methods require further investigation