NMR visibility studies of N‐δ proton of proximal histidine in deoxyhemoglobin in lysed and intact red cells

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Abstract
Recent in vivo1H MRS studies of muscle oxygen levels by means of myoglobin (Mb) oxygenation have necessitated an investigation of the possible contamination from deoxy‐hemoglobin (Hb). To determine the contribution of Hb in these NMR measurements, we studied the NMR visibility of histidyl N‐δ proton of Hb in lysed and intact RBC (red blood cell) suspensions. We found that the visibility of deoxy‐Hb was significantly lower in intact RBCs (ca. 16%) than the solution form at an equivalent concentration of β heme‐iron, even though the visibility of deoxy‐Hb from lysed RBC and deoxy‐Mb was almost identical. Our study also shows that visibility increases with RBC swelling. Based on the difference in concentration and compartmentation, we conclude that less than 17% contribution from Hb is expected in Mb measurements in vivo. The possible mechanisms which cause the low visibility of Hb in RBCs are discussed.