Dielectric Response of Cytoplasmic Water and Its Connection to the

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The Dielectric Response of Cytoplasmic Water and Its Connection to the Vitality of Human Red Blood Cells: I. the Glucose Concentration Influence Evgeniya Levy, Gregory Barshtein, Leonid Livshits, Paul Ben Ishai, and Yuri Feldman J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b06996 • Publication Date (Web): 12 Sep 2016 Downloaded from http://pubs.acs.org on September 16, 2016

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The Journal of Physical Chemistry

The Dielectric Response of Cytoplasmic Water and its Connection to the Vitality of Human Red Blood Cells: I. The Glucose Concentration Influence Evgeniya Levy†, Gregory Barshtein‡, Leonid Livshits‡, Paul Ben Ishai†§, and Yuri Feldman*,† †

Department of Applied Physics, The Rachel and Selim Benin School of Engineering and Computer Science, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Jerusalem, 91904, Israel ‡ Department of Biochemistry & Molecular Biology, IMRIC, Faculty of Medicine, The Hebrew University of Jerusalem, Ein Kerem, Jerusalem, 91120, Israel § Department of Physics, Ariel University, P.O.B. 3, Ariel 40700, Israel

ABSTRACT The vitality of red blood cells depends on the process control of glucose homeostasis, including the membrane’s ability to “switch off” D-glucose uptake at the physiologically specific concentration of 10 – 12 mM. We present a comprehensive study of human erythrocytes suspended in buffer solutions with varying concentrations of D-glucose at room temperature, using microwave dielectric spectroscopy (0.5 GHz – 50 GHz) and cell deformability characterization (the Elongation ratio). By use of mixture formulas the contribution of the cytoplasm to the dielectric spectra was isolated. It reveals a strong dependence on the concentration of buffer D-Glucose. Tellingly, the concentration 10 – 12 mM is revealed as a critical point in the behavior. The dielectric response of cytoplasm depends on dipole-matrix interactions between water structures and moieties like ATP, produced during glycolysis. Subsequently, it is a marker of cellular health. One would hope that this mechanism could provide a new vista on non-invasive glucose monitoring.

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INTRODUCTION Red blood cells (RBCs), also called erythrocytes, are the most common and abundant type of blood cells. They are non-spherical biconcave discs and consist of a cellular membrane and a cytoplasm that is rich in hemoglobin. Their task is to transport oxygen in and carbon dioxide out of the organism. The plasma membrane of erythrocytes is nonconductive compared to their cytoplasm, which results in charge accumulation and interfacial polarization. This leads to the well-known β-dispersion in RBC dielectric spectrum at the 0.l – 10 MHz1. This dispersion has been studied intensively from the beginning of the twentieth century and has resulted in some intriguing conclusions. For example, using the dielectric properties of β-dispersion, the molecular thickness of plasma membrane was estimated1. Since then, the electrical properties of RBC have been extensively investigated2-11 and the electrical parameters (the membrane permittivity and the membrane conductivity) can be considered to be well established. Glucose is essential for the function of erythrocytes12. Recently, it was shown6-7,

9

that the

membrane capacitance (or the membrane permittivity) is altered by the presence of D-glucose and dependent on its concentration in the buffer. Furthermore, the membrane capacitance is also linked to the geometric shape the cells. In the case of spherical cells (in hypotonic saline solution) this dependence is non monotonic in its behavior, with a critical point close to 12 mM D-glucose concentration. Additionally, it has been reported that erythrocyte ghosts (the plasma membrane of an erythrocyte void of hemoglobin) in phosphate buffered saline (PBS), change back to a biconcave shape with increasing of adenosine triphosphate (ATP) concentration in the suspension13. This indicates that ATP interaction with the cytoskeleton is responsible for cellular shape. In erythrocytes, ATP is mainly produced by anaerobic glycolysis, following the uptake of glucose through the membrane glucose transporter GLUT11, 13-14. The key regulative processes


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of glucose penetration are also dependent on intracellular ATP and Adenosine monophosphate (AMP) and how they bind to this membrane protein13-14. Recently to measure blood glucose levels non-invasively by using microwave sensors attached to the skin. The dielectric response of these sensors, usually operating in the high GHz frequency region, has shown some sensitivity to the presence of glucose in the blood15-23. Clearly, at these frequencies this is not related to any interfacial polarization effect, due to the cell membrane, but rather to the dielectric response of water. Consequently, the role of water in the monitoring of glucose by using microwave frequencies appears to be extremely important. The RBC cytoplasm can be considered as a concentrated aqueous solution with its main relaxation peak at dozens of gigahertz. It is well known that over a wide temperature range and at frequencies up to 40 GHz, the experimental complex permittivity spectra of the bulk water can be described by the simple Debye function24-25. However, whenever water interacts with another dipolar or charged entity, a symmetrical broadening of its dispersion peak and a change in the attendant relaxation time26-30 is induced. The origin of the alteration of the dielectric loss peak is defined by the dynamics of H-bond network rearrangements in the vicinity of the different solute molecules. The modification of water’s relaxation peak can be described by the phenomenological Cole-Cole (CC) function31: ∆

ε∗ = ε − ε" = + ,

(1)

Here ε′ and ε″ are the real and the imaginary parts of the complex permittivity, ω = 2πf is the cyclic frequency, and i2 = -1. The parameter denotes the extrapolated high-frequency permittivity and ∆ = − , is the relaxation amplitude (with the low frequency permittivity limit denoted by ). The exponent α (0 < α ≤ 1) is a measure of the symmetrical broadening. It was shown that the relationship between the parameters of the main peak of water in aqueous 3 ACS Paragon Plus Environment


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solutions is linked to the type of the solute and can be combined in a new phenomenological approach26,

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to describe the dynamics of the water/solute interaction. There exists a

fundamental connection between the relaxation time, , the broadening parameter, , and the Kirkwood-Froehlich correlation function , itself dependent on Δ (see Appendix A). This approach has been applied to some simple aqueous solutions27-30 where the solute was either dipolar or ionic. In these systems we were able to verify the specific mechanism of solute (matrix) interaction with water dipoles and illuminate the differences between events that are controlled by either dipole-dipole or ion-dipole interactions. These findings shed light on the mechanism of hydration shell formation in each case. The CC parameters of the main water peak can be considered as markers that can help to clarify the type (ion-dipole or dipole-dipole) and the rate at which water interacts with a solute33. Here we will study the microwave dielectric response of water in the cytoplasm of erythrocytes subjected to a wide range of glucose concentrations in the buffer. These results will be supplemented by direct measurements of cell deformability34-35. The approach described above will then be used to offer new insights to the metabolism of RBC.

MATERIALS AND METHODS Cells preparation The cells preparation protocol was based on the protocol detailed in ref.7. Three human blood samples were collected from healthy donors (upon their consent under the Helsinki Ethics Committee (#0568-12-HMO), Hadassah Hospital, Israel). These samples were drained to plastic vacutainer (BD Vacutainer® PST™) with lithium heparin (68 I.U.). RBC were isolated from the blood by centrifugation, washed (twice) from their plasma, once again by centrifugation (500 x g for 10 min), in PBS, and re-suspended at a hematocrit of about 15% in PBS (pH 7.4). This 4 ACS Paragon Plus Environment

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routine preserves the biconcave shape of the cells. The concentration of RBC in suspension was controlled by a complete blood counter (Automated Hematology Analyzer, XP-300, Symex America, Inc, USA). The washed and isolated erythrocytes were then re-suspended in PBS and supplied with nine differing D-glucose (Sigma-Aldrich G5400) concentrations ranging from 0 to 20 mM (i.e., 0, 2, 5, 8, 10, 12, 15, 18 and 20 mM). In order to keep the final osmolarity of the suspensions constant, a correspondent concentration of L-glucose (Sigma-Aldrich G5500) had to be added to the PBS buffer for each concentration batch. L-glucose is an optic enantiomer of Dglucose that is not transportable through the erythrocyte membrane and is frequently used as a control in glucose transport and kinetic studies7.

Dielectric spectroscopy Dielectric measurements were carried out in the frequency range from 500 MHz to 50 GHz using a Microwave Network Analyzer (Keysight N5245A PNA-X), together with a Flexible Cable and Slim-Form Probe (Keysight N1501A Dielectric Probe Kit). The calibration of the system was performed with the aid of three references: air, a Keysight standard short circuit and pure water at 25°C. A special stand for the Slim-Form Probe was designed and combined with a sample cell holder for liquids (total volume ~ 7.8mL). The holder was enveloped by a thermal jacket and attached to a Julabo CF 41 oil-based heat circulatory system. The cell was held at 25°C by the circulator-thermostat with temperature fluctuations less than 0.1°C. The whole measuring system was placed in an air-conditioned room maintained at 25 ± 1°C. Each sample was measured at least six times. The real and imaginary parts ε'(ω) and ε''(ω) were evaluated using the Keysight N1500A

Materials

Measurement

Software

with

an

accuracy

of

∆ ′ ⁄ ′ = 0.05,

∆ ′′ ⁄ ′′ = 0.0536.



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When D-glucose was added to the PBS, the cells were first incubated for 5 minutes at 25 °C before dielectric measurements were made.

Determination of RBC deformability The present research employed the computerized Cell Flow-Properties Analyzer (CFA), designed and constructed in house34. The CFA enables the monitoring of RBC hemodynamic characteristics as a function of shear stress, under conditions resembling those in micro-vessels, by a direct visualization of their dynamic organization in a narrow-gap flow-chamber that has been placed under a microscope34,

37-38

. RBC deformability is determined by monitoring the

elongation of RBCs, while they are stuck to a polystyrene slide, under flow-induced sheer stress 34

. In brief, 50 µl of RBC suspension (1% hematocrit, in PBS) are inserted into the flow-chamber

(adjusted to 200 µm gap) containing an un-coated slide (purchased from Electron Microscopy Science (Washington, PA)). The RBCs that adhere to the slide surface are then subjected to controllable flow-induced sheer stress (3.0 Pa), and their deformability is determined by the change in cell shape. This change is expressed by the elongation ratio: ER = a/b, where a is the major cellular axis and b is the minor cellular axis. ER = 1 reflects a round RBC, undeformed by the applied sheer stress. The CFA contains an image analysis program capable of automatically measuring the ER for individual cells. The deformability distribution of a large RBC population (at least 2500 ± 300 cells) is then provided as a function of shear stress34. The accuracy of the axes measurement is about 10%. RBCs with ER ≤ 1.1 are defined as “undeformable” cells, namely the cells that do not deform under the high sheer stress, 3.0 Pa, used in this study.

RESULTS AND DISCUSSION An example of typical dielectric loss spectra for a RBC suspension and pure water at 250C are presented in the Figure 1a. The dc conductivity contribution has been removed from the spectra. 6 ACS Paragon Plus Environment

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30

20

Losses, ε"

Losses, ε"

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20

10

10

0

(a)

1

Frequency, f [GHz]

10

0

(b)

1

Frequency, f [GHz]

10

Figure 1. In panel (a) are shown the dielectric loss spectra at 25 °C of an RBC suspension in PBS with 0 mM D-glucose (red triangles) compared to water (black line). Dc conductivity has been removed. In panel (b) are shown the derived losses of cellular cytoplasm for 0 mM D-glucose (red triangles) and 10mM D-glucose (blue circles). The lines are the fitting curves

At the frequencies measured, the plasma membrane of the cell is transparent, and therefore the relative permittivity of the cytoplasm can be obtained from those of the cell suspension using an appropriate mixture equation39-40. The optimal model in this case was first presented by Kraszewski41, who considered the microwave propagation in a heterogeneous medium. The underlying assumption is that a biphasic suspension can be considered as a sum of an infinite number of thin water and substance layers, each of thickness δt

Dielectric Response of Cytoplasmic Water and Its Connection to the

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