Determination of Methemoglobin in Hemoglobin Submicron Particles Using NMR Relaxometry.

Waraporn Kaewprayoon,Chiraphat Kloypan,Nittiya Suwannasom,Axel Steffen,Yu Xiong,Axel Pruß,Radostina Georgieva,Eyk Schellenberger,Hans Bäumler

doi:10.3390/ijms21238978

Abstract

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.

Highlights

Hemoglobin (Hb), the main component of red blood cells (RBC), is an iron-containing oxygen-transporting metalloprotein
The relaxation rates 1/T1 and 1/T2 (Figure 2A,B, respectively) of RBC suspensions cross-linked with four different GA concentrations are plotted against the total Hb concentration in the samples
The treatment with NaNO2 resulted in particles with more than 96% MetHb

Summary

Introduction

Hemoglobin (Hb), the main component of red blood cells (RBC), is an iron-containing oxygen-transporting metalloprotein. The relaxation rates 1/T1 and 1/T2 (Figure 2A,B, respectively) of RBC suspensions cross-linked with four different GA concentrations are plotted against the total Hb concentration (cHb) in the samples. Transverse relaxation rate (1/T2), respectively, of HSA-MP suspensions prepared with different concentrations of GA depending on the particle volume concentration (packed particle volume, PPV). The transverse relaxivity, r2, of MetHb-MP is not very different from that of the Hb-MP, because the cross-linking of the proteins is completed and the paramagnetic effect of MetHb has no influence on T2. The results obtained for suspensions of RBCs cross-linked with different GA concentrations revealed that the longitudinal and the transverse relaxation behavior deliver information on the two different aspects of the GA cross-linking: (i) degree of iron oxidation and MetHb generation via longitudinal relaxivity r1 and (ii) degree of cross-linking and protein network formation via transverse relaxivity r2, respectively. 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

Materials

Preparation of Hb and MetHb Solutions

Determination of Oxyhemoglobin in RBC Suspensions and Hb Solutions

Findings

NMR Analysis