Introduction
The magnetic properties of hemoglobin(Hb) and its derivative forms, have been documented since the 1930’s (Pauling & Coryell, 1936; Pauling & Coryell, 1936), and since 2010, there have been at least 92 publications related to the magnetic separation of red blood cells (RBCs) based on their intracellular Hb content. Many groups are investigating label-free methods to remove RBCs from whole blood by exploiting the diamagnetic properties of white blood cells and the paramagnetic properties of RBCs when the Hb is in its deoxy or met form (Furlani 2007; Han & Frazier, 2005; Han & Frazier, 2006; Fattah, Ghosh & Puri, 2016; Moore et al. , 2006; Norina, Shalygin & Rastopov, 2000; Wu et al. , 2016; Kim, Massoudi, Antaki & Gandini, 2012; Kashevsky, Zholud & Kashevsky, 2015; Nam, Huang, Lim, Lim & Shin, 2013; Kawano & Watarai, 2012; Shen, Hwang, Hahn & Park, 2012; Jung, Choi & Han, 2010; Zborowski et al. , 2003; Moore et al. , 2018). The magnetic susceptibilities of oxygenated (oxyRBCs), deoxygenated (deoxyRBCs) and methemoglobin-RBCs (metRBCs) were originally predicted (Pauling & Coryell, 1936; Pauling & Coryell, 1936; Zborowski et al. , 2003; Spees, Yablonskiy, Oswood & Ackerman, 2001) from theoretical calculations and subsequently validated experimentally, with a slightly greater magnetic susceptibility for metRBCs when compared to deoxyRBCs. Although the magnetic properties of deoxyRBCs and metRBCs are well studied, the existing methods to deoxygenate RBCs in a reversible manner are complex. Thus, novel strategies to turn oxyRBCs into the magnetic deoxyRBCs are desired for blood taken from both healthy donors and patients suffering from hematological disorders.
In the studies to be presented below, human blood from normal and sickle cell disease, SCD, donors was used. SCD is a monogenic disorder where the patient is a heterozygous or homozygous carrier for the βS allele. This mutation causes a single nucleotide substitution from glutamic acid to valine in the β-globin chain and the resulting β6 substitution results in hydrophobic interactions with adjacent HbS (sickle cell hemoglobin) chains. This polymerization is triggered at low pO2 and is also dependent on intracellular HbS and HbF (fetal hemoglobin) and pH. Studying how SCD Hb is influenced by these parameters potentially makes possible new diagnostic and therapeutic techniques. One specific need is a rapid test to diagnose a SCD “crisis”, in which rigid, sickled erythrocytes adhere to the endothelium and cause vaso-occlusion. A potential therapeutic example of the application of difference in magnetic properties of RBCs would be the return to the patient of normal RBCs during an exchange transfusion where plasma is returned to the patient with an apheresis device and all of the removed RBCs are discarded. It is this discarded apheresis RBC product that is used in this present study (Bunn, 1997; Rees, Williams & Glad, 2010; Stuart & Nagel, 2004).
The most commonly used chemical reaction to yield metRBCs, the magnetic form of RBCs, is the addition of sodium nitrite to oxyHb RBCs (Doyle, Pickering, DeWeert, Hoekstra & Pater, 1981; Winterbourn, 1985; Norina, Shalygin & Rastopov, 2000; Nam, Huang, Lim, Lim & Shin, 2013; Zborowski et al., 2003) in a simple, single step and has a conversion near 100% (Winterbourn, 1985). This metHb is stable after several washing steps and is preferred to mimic the paramagnetic properties of deoxyRBCs due to its ease of preparation. However, metHb is not a realistic candidate for subsequent RBC use, such as fractionation studies of high and low content of Hb within RBCs, since the metHb loses its ability to bind with, and release oxygen (methemoglobinemia). The globin structure destabilizes upon metHb conversion to hemichrome when reacted with H2O2 or diazine. MetHb can react with semiquinones to form oxyHb once again, but the formation of MetHb is stable over a long period of time and is irreversible for practical uses such as paramagnetic RBCs separation with subsequent use of the RBCs. Winterbourn (1990) and Di Iorio (1981) prepared MetHb using superoxide or potassium ferricyanide but these methods are much less popular compared to using sodium nitrite. Additionally, the reaction between deoxyHb and NaNO2 produces the vasodilator NO and nitrosylated hemoglobin (Cosby et al. , 2003).
Methods to prepare deoxygenated RBCs, however, are much more complex. Several reports exist of using either bubbled N2, Ar, or another oxygen-free inert gas through the blood or applying a gentle vacuum (Benesch, Benesch, Renthal & Maeda, 1972; Zijlstra, Buursma & Meeuwsen-van der Roest, 1991; Kawano & Watarai, 2012; Zborowskiet al. , 2003), add sodium dithionite (Moore, Schygulla, Strohm & Kolios, 2016; Han & Frazier, 2005; Shen, Hwang, Hahn & Park, 2012; Jung, Choi, Han & Frazier, 2010), or both methods at once (Lambeth & Palmer, 1973; Azarov et al. , 2005; Hofrichter, Ross & Eaton, 1974; Eaton, Hanson, Stephens, Sutherland & Dunn, 1978). Moore et al. (2018) used a hollow fiber deoxygenator, but methods, such as this involving gas exchange, must keep the system sealed from the atmosphere. In order to characterize the hemoglobin, absorbance spectra of anaerobic samples must be measured using stopped-flow spectrophotometers to keep the sample free of oxygen. This equipment is more complex compared to basic spectrophotometers. Stopped-flow spectrophotometric measurements can be made directly on RBCs in a diffusion-limited reaction (Coin & Olson, 1979) or directly to hemoglobin obtained from cell lysis. The oxygenation kinetics of deoxyRBCs and free deoxyHb are discussed in Coin & Olson (1979) and several other studies have described kinetic models and rate constants for other reactions involving derivatives of hemoglobin (Antonini & Gibson, 1960; Azarov et al. , 2005; Doyle, Pickering, DeWeert, Hoekstra & Pater, 1981). These studies use stopped-flow spectrophotometers and extinction coefficients to calculate the concentrations of other forms of hemoglobin.
Another method to obtain deoxyHb involves the use of sodium dithionite. In an aqueous solution, sodium dithionite reacts with oxygen and water to produce sodium bisulfite and hydrogen peroxide. If used in dilute hemoglobin concentrations, hydrogen peroxide, and subsequently free radicals, become more abundant. In the solid phase, sodium dithionite will react with ambient oxygen to produce sulfite and thiosulfate, making weight measurement inaccurate. The actual amount dissolved into a sample must be determined from titration with a standard ferricyanide solution and following reduction at 420nm (or measuring absorption at 314nm, which is less accurate). Buffers for sodium dithionite must also be above pH 7.6 due to instability. Lastly, dithionite has been shown to alter the isosbestic point between oxyHb, deoxyHb and carboxyHb (Di Iorio, 1981; Mook, Buursma, Gerding, Kwant & Zijlstra, 1979; Zijlstra, Buursma & Meeuwsen-van der Roest, 1991). These points make sodium dithionite experimentally inaccurate and risky in clinical settings.
In contrast, and to the best of our knowledge, no reports exist on the use of the commercial enzyme product, EC-Oxyrase® (referred to as Oxyrase moving forward, not to be confused with the company name), to deoxygenate RBCs. In brief, Oxyrase name was given to an enzyme that allows radiation-damaged cells to divide, without the enzyme entering or damaging the cell. With addition of a proton donor, the enzyme consumes dissolved oxygen and converts it to water in media deoxygenating the media without airtight containers or flushing the solution with inert gases (Adler & Spady, 1997). The rate of DO removal (activity of Oxyrase), is highly pH dependent, with a maximum activity at pH 8.4 and 55°C (“EC Oxyrase”, 2019). Other uses of Oxyrase include growing anaerobic cells, increasing cell growth in the log phase, increasing maximum titer and protecting certain biomolecules from reactions with oxygen.
The active components of Oxyrase have been shown to repair damaged cells, accelerate cell growth and consume dissolved oxygen. These enzymes, which also originate from the cytoplasmic membrane fraction ofE. Coli , have high reactivity with NADH and succinate (Adler, Carrasco, Crow & Gill, 1981). These processes are due to active electron transport chain, ETC, complexes by which ATP is produced from NADH or succinate and H+ ions from within the cell. Mechanisms in eukaryotic and prokaryotic are identical, however they take place in different domains. Eukaryotic ETC takes place across the inner membrane of the mitochondria, between the matrix and inner membrane space while still within the outer membrane of the cell. Prokaryotic ETC takes place across the membrane, between the cytoplasm and outside of the cell. Five complexes (CI, CII, CIII, CIV, CV) are involved. Briefly, CI and CII catalyze the feed molecule (NADH or succinate for CI and CII, respectively) into ubiquinone (QH2) and transport the product to CIII. CIII and CIV are cytochromes, which contain active heme molecules (6 for CIII and 2 for CIV). CIII transports 2H+ to CIV via cytochrome c. Cytochrome c is a mobile electron carrier, while the transport chain from CI or CII to CIII occurs within a supercomplex of CI-CIII-CIV or CII-CIII-CIV. CIV reduces dissolved oxygen with the ions from cytochrome c to produce H2O. (Fisher, Adler, Shull & Cohen, 1969; Guo, Gu, Zong, Wu & Yang, 2018; Zhao, Jiang & Zhang, 2011)
This study evaluates the performance of Oxyrase to remove oxygen from intracellular and free hemoglobin; thus resulting in changes in the magnetic susceptibility of hemoglobin, and correspondingly when the hemoglobin is in RBC, the RBC magnetic susceptibility. The results from the use of an Eppendorf BioSpectrometer Basic, and the Winterbourn equation (Winterbourn, 1990) and Cell Tracking Velocimetry, (presented in detail in Zborowski et al. (2003), Moore et al. (2006), Moore et al. (2000) and Zhang et al. (2005)) were used to experimentally quantify the deoxygenation of suspended Hb and Hb retained within RBCs, respectively. The motivation for this current study is to develop a convenient, easily reversible, nontoxic, single step deoxygenation of red cells. Future studies seek to separate RBC samples based on iron content and re-transfuse.