Dielectric Responses of Cytosolic Water Change with Aging of Circulating Red Blood Cells

Abstract

Water molecules in the cytosol of red blood cells (RBCs) may exist in a free or bound state. The ratio between the free and bound water depends on the composition of the cytoplasm, particularly on the hemoglobin concentration. Microwave dielectric spectroscopy (MDS) provides information on the state of intracellular water in red blood cell suspension and the erythrocyte cytosol state. In the presented study, we used MDS to assess the differences in the free-to-bound water ratio in subpopulations of freshly donated human erythrocytes of different ages (young, mature, and senescent cells) obtained by fractionation in a Percoll density gradient. The obtained MDS parameters (dielectric strength ∆ε, the relaxation time τ, and the broadening parameter α) were compared with the red blood cell indices and single cell deformability measurements obtained for each subpopulation. We demonstrated that the unique hematological indices and deformability of red blood cells of different ages are well-correlated with the specific values of dielectric fitting parameters. The obtained results indicate that the dielectric properties of cytosolic water can serve as a sensitive marker of changes occurring in the cytosol of red blood cells during cell aging.

Keywords: Percoll gradient; RBC aging; hematological indices; microwave dielectric spectroscopy; red blood cells.

Figure 1

Dielectric spectra of the young RBC suspension (orange line) and the old RBC suspension (green line) compared to phosphate buffered saline–PBS (red line) and distilled water (blue line). The real part of the static permittivity (ε′) is lower in the case of the RBC suspensions (area 1). Additionally, the imaginary part (ε″) of the RBC suspensions is characterized by the shift and broadening of the main relaxation peak (area 2) and the appearance of the conductivity tail (area 3). The hematocrit was 15% for both young and old RBC suspensions.

Figure 2

(A) The real part of the dielectric permittivity (ε′) and (B) dielectric losses (ε″) of the cytoplasmic water of young RBCs (orange) and old RBCs (from the same donor). The cytosolic water was extracted using the Kraszewski mixture formula (see Equation (2)). The low-frequency regions in the dielectric loss spectra (B) are omitted, as they only represent ionic contributions. This study focuses on the state of bulk water in the system, which is characterized by the main dispersion peak at higher frequencies.

Figure 4

Deformability of RBCs from three density fractions. (A) Typical images of RBCs deformation and relevant distribution (B) of elongation ratio (ER) in RBCs population for cells from Y-, M-, and O-fractions. Shear stress is 3.0 Pa. Image size is 150 × 100 μm.

Figure 3

Fitting parameters for Y-, M-, and O-fractions of RBCs for four healthy donors. Each datum is Mean ± SE obtained from six measurements. Statistical analysis of data is presented in Table S3; Supplementary Materials).

Figure 5

MER for the three density fractions (Y, M, and O) of erythrocytes. Each value represents the mean value ± standard error obtained from the measurements of six healthy donors. The significance of the difference in the MER value is shown for the M- and O-fractions compared to the young fraction. p—*, 0.01 and **, 0.0004.

Figure 6

Correlation between dielectric fitting parameters and MCHC for three RBCs fractions: young (black), mature (red), and old (green). Each date was presented as mean ± SE from four samples.

Figure 7

Correlation of the dielectric strength (∆ε) with the MCHC (left, R² = 0.553; p = 0.00017) and the MCV (right, R² = 0.613; p = 0.00005) for four healthy donors.

Figure 8

Correlation between dielectric fitting parameters and %UDFCs for three RBCs fractions: young (black), mature (red), and old fraction (green). Each date was presented as mean ± SE from four samples.

Figure 9

Correlation between dielectric strength (∆ε) with MER (left, R² = 0.306; p = 0.014) and % UDFCs (right, R² = 0.330; p = 0.01) for four healthy donors.