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.