International Journal of Molecular Sciences Article Determination of Methemoglobin in Hemoglobin Submicron Particles Using NMR Relaxometry Waraporn Kaewprayoon 1,2, Nittiya Suwannasom 1,3 , Chiraphat Kloypan 1,4 , Axel Steffen 1 , Yu Xiong 1 , Eyk Schellenberger 5, Axel Pruß 1, Radostina Georgieva 1,6 and Hans Bäumler 1,* 1 Charité-Universitätsmedizin Berlin, Institute of Transfusion Medicine, 10117 Berlin, Germany; [email protected] (W.K.); [email protected] (N.S.); [email protected] (C.K.); Axel.steff[email protected] (A.S.); [email protected] (Y.X.); [email protected] (A.P.); [email protected] (R.G.) 2 Department of Pharmacy, Payap University, Chiang Mai 50000, Thailand 3 Division of Biochemistry and Nutrition, School of Medical Sciences, University of Phayao, Phayao 56000, Thailand 4 Division of Clinical Immunology and Transfusion Sciences, School of Allied Health Sciences, University of Phayao, Phayao 56000, Thailand 5 Charité-Universitätsmedizin Berlin, Institute of Radiology and Children Radiology, 10117 Berlin, Germany; [email protected] 6 Department of Medical Physics, Biophysics and Radiology, Faculty of Medicine, Trakia University, 6000 Stara Zagora, Bulgaria * Correspondence: [email protected]; Tel.: +49-(30)-45052-5131 Received: 30 October 2020; Accepted: 23 November 2020; Published: 26 November 2020 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 1 spectrophotometry is not possible. Here, we demonstrate that H2O 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. 1 Keywords: methemoglobin; hemoglobin microparticles (Hb-MP); H2O NMR-relaxometry; hemoglobin-based oxygen carriers 1. Introduction Hemoglobin (Hb), the main component of red blood cells (RBC), is an iron-containing oxygen-transporting metalloprotein. It contains four heme groups each incorporating an iron atom in 2+ the ferrous state (Fe ), which can reversibly bind molecular oxygen (O2), resulting in two states of Hb—the oxygenated Hb (OxyHb) and the deoxygenated Hb (DeoxyHb). In this way, RBC are loaded with O2 in the lung (OxyHb) and O2 is delivered to the organs/tissue (DeoxyHb) [1,2]. In contrast Int. J. Mol. Sci. 2020, 21, 8978; doi:10.3390/ijms21238978 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 8978 2 of 13 to these two Hb-states, Hb derivatives are altered forms of hemoglobin, which are formed by the combination of different iron ligands in the heme or by the oxidation state of the iron. After oxidation, the iron in the heme group changes to the ferric state (Fe3+) and Hb becomes methemoglobin (MetHb). MetHb cannot bind or carry oxygen molecules since the sixth coordination position of iron is bound either to a water molecule or a hydroxyl group [3,4]. Furthermore, the presence of MetHb increases the affinity of the remaining Hb or heme groups to oxygen, which makes the oxygen release in the tissues more difficult [4,5]. Normally one to two percent of a person’s Hb is MetHb [5]. A higher percentage, can be of genetic origin or caused by exposure to various chemicals [3,6]. Depending on the level of MetHb in the blood, it can cause health problems known as methemoglobinemia. Therefore, the MetHb concentration in blood is a very important diagnostic parameter in cases of hypoxia. One of common methods of hemoglobin analysis in clinical laboratories are based on the so called multiple wavelength spectrophotometry (CO-oximetry) [7–10], which exploits the differences in the absorption spectra of OxyHb, DeoxyHb, and MetHb. Before measurement, Hb is released from the RBCs by hemolysis to avoid the light scattering by the cells. Recently, novel hemoglobin microparticles (Hb-MP) have been developed and proposed as artificial oxygen carriers. The fabrication procedure was published under the abbreviation CCD-technique (Co-precipitation-Crosslinking-Dissolution) [11–13] that reflects the three main preparation steps: co-precipitation of Hb with MnCO3, crosslinking of Hb and dissolution of the MnCO3. The resulting particles consist of cross-linked Hb and show a narrow size distribution around 700 nm. The particles were investigated spectrophotometrically to demonstrate their oxygenation and deoxygenation [11,14]. However, Hb-MP cannot be lysed like RBC and a quantitative determination of OxyHb, DeoxyHb, and MetHb by multiple-wavelength spectrophotometry is not possible due to the strong light scattering of the Hb-MP suspensions. It is known, that the cross-linking of Hb by glutaraldehyde (GA) causes a certain amount of MetHb to arise [15]. Therefore, a quantitative measurement of the MetHb content of the Hb-MP is very important in view of their potential application as an artificial blood substitute and alternatives to spectrophotometric measurements are needed. In previous studies, the oxygen carrying capacity of the Hb-MP was used for the indirect determination of dyshemoglobin or nonfunctional hemoglobin. The oxygen released after oxidation of the Hb by cyanide was directly measured and related to the amount of functional Hb in the Hb-MP [12,16]. The difference between total Hb and functional Hb equals nonfunctional hemoglobin. However, this method does not allow a direct measurement of MetHb and presupposes 100% oxygenation of the Hb in the Hb-MP. 1 The water proton nuclear magnetic resonance ( H2O NMR) relaxometry could be a suitable method that can be used for quantification of the MetHb concentration directly by measuring the oxidative state of iron in the Hb-MP.The different oxidation state of the iron in MetHb means that MetHb has a different electronic configuration and, therefore, different paramagnetic properties compared to 1 that of Hb [17]. This difference can be detected using NMR spectroscopy by measuring the H2O NMR longitudinal relaxation time (T1) and the transverse relaxation time (T2) or the corresponding relaxation rates (1/T1 and 1/T2). Previous studies on methemoglobin concentrations in blood samples have shown comparable values obtained by NMR and multiples-wavelength spectrophotometry [18,19] but the NMR method is not influenced by light scattering and does not require lysis of RBC. Additionally, NMR provides information on the molecular environment and therefore it would allow to investigate the influence of the GA crosslinking on the molecular structure of Hb in the Hb-MP. 1 The aim of this study was to demonstrate that the H2O NMR relaxation rates can be used to determine the total Hb as well as MetHb content of Hb-MP, because multiple wavelength 1 spectrophotometry cannot be applied. Therefore, the H2O NMR relaxation times of Hb in Hb-solutions and in vital RBC were measured at various MetHb concentrations to investigate the influence of different surroundings on their relaxation properties. The influence of the GA crosslinking on the 1 H2O NMR relaxation rates was investigated using GA cross-linked RBC and GA HSA-MP. Finally, Int. J. Mol. Sci. 2020, 21, 8978 3 of 13 1 Hb-MP suspensions with different MetHb contents were prepared and the H2O NMR relaxation rates wereInt. J. measured.Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 14 2.2. ResultsResults andand DiscussionDiscussion 2.1.2.1. NMRNMR ofof HumanHuman HbHb/MetHb/MetHb Solutions Solutions and and RBCs RBCs/MetHb-RBC/MetHb-RBC Suspensions Suspensions FigureFigure1 1shows shows thethe relaxationrelaxation rates 1/T 1 /T1 and1 and 1/T 12/ Tof2 Hbof Hbsolutions solutions and andsuspensions suspensions of intact of intact RBCs RBCsdepending depending on the on MetHb the MetHb concentration. concentration. It can It be can seen be seenthat thatboth, both, the thelongitudinal longitudinal as well as well as the as thetransverse transverse relaxation relaxation accelerate accelerate with with increasing increasing MetHb MetHb concentration. concentration. The The increase ofof 11/T/T11 withwith increasingincreasing MetHb MetHb concentration concentration is is roughly roughly equivalent equivalent in in Hb Hb solutions solutions and and suspensions suspensions of of intact intact RBCs RBCs (Figure(Figure1 A),1A), whereas whereas 1 1/T/T2 2increases increases much much faster faster with with the the MetHb MetHb concentration concentration in in RBC RBC suspensions suspensions (Figure(Figure1 B).1B). This This is is in in agreement agreement with with the th resultse results obtained obtained by by other other groups groups [20 [20–23].–23]. Figure 1. Relaxation rates 1/T1 (A) and 1/T2 (B) of Hb solutions and of RBC suspension as a function of Figure 1. Relaxation rates 1/T1 (A) and 1/T2 (B) of Hb solutions and of RBC suspension as a function the MetHb concentration (measured at proton frequency 40 MHz and temperature 37 C). The values of the MetHb concentration (measured at proton frequency 40 MHz and temperature◦ 37 °C). The represent means and standard deviations of N = 6 samples. The slopes of 1/T1 for Hb solutions (0.0226) values represent means and standard deviations of N = 6 samples.