Determination of Methemoglobin in Hemoglobin Submicron Particles Using NMR Relaxometry

Methemoglobin (MetHb) is a hemoglobin (Hb) derivative with the heme iron in ferric state (Fe³⁺), unable to deliver oxygen. Quantification of methemoglobin is a very important diagnostic parameter in hypoxia. Recently, novel hemoglobin microparticles (Hb-MP) with a narrow size distribution around 700 nm, consisting of cross-linked Hb were proposed as artificial oxygen carriers. The cross-linking of Hb by glutaraldehyde (GA) generates a certain amount of MetHb. Due to the strong light scattering, quantitative determination of MetHb in Hb-MP suspensions by common spectrophotometry is not possible. Here, we demonstrate that ¹H₂O NMR relaxometry is a perfect tool for direct measurement of total Hb and MetHb concentrations in Hb-MP samples. The longitudinal relaxation rate 1/T₁ shows a linear increase with increasing MetHb concentration, whereas the transverse relaxation rate 1/T₂ linearly increases with the total Hb concentration. In both linear regressions the determination coefficient (R²) is higher than 0.99. The method does not require time-consuming pretreatment or digestion of the particles and is not impaired by light scattering. Therefore, it can be established as the method of choice for the quality control of Hb-MP and similar hemoglobin-based oxygen carriers in the future.