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6STUDY OF CHANGES IN THE OPTICAL CHARACTERISTICS OF BLOOD COMPONENTS DURING THE
DEVELOPMENT OF MODEL ALLOXAN DIABETES IN RATS USING REFRACTOMETRY AND
FLUORESCENCE SPECTROSCOPY
E. N. LAZAREVA u*, N. A. SHUSHUNOVA1, A. B. BUCHARSKAYA2,3, V.I. KOCHUBEY1, V. V. TUCHIN1,2,4
'Science Medical Center, Saratov State University, 83 Astrakhanskaya str., 410012 Saratov, Russia
2Laboratory of Laser Molecular Imaging and Machine Learning, Tomsk State University, 36 Lenin av., 634050 Tomsk,
Russia
3Saratov State Medical University named after V.I. Razumovsky, Saratov, Russia
4Laboratory of Laser Diagnostics of Technical and Living Systems, Institute of Precision Mechanics and Control of the
RAS, 24 Rabochaya str., 410028 Saratov, Russia
*email address: lazarevaen@ list.ru
ABSTRACT
Among the diagnostic methods, there are methods based on the quantitative determination of the concentration of glycated proteins, which are biological markers of the disease [1-3]. One of them, which is often used as a marker, is HbA1c, which is formed as a result of non-enzymatic glycation of hemoglobin, which is exposed to free glucose in the blood and, therefore, has a strong correlation with the average concentration of glucose in the blood in the preceding three-month period. Because of this strong correlation, HbA1c levels were regularly used for monitoring long-term glucose control in established diabetics and recently approved for screening for diabetes (HbAlc > 6.5%) and prediabetes (5.7% < HbAlc < 6.4%) [4 -8].
Currently, there are more than 20 methods for determining glycated hemoglobin, such as cation-exchange chromatography, electrophoresis, affinity chromatography and immunoanalysis, but each of them measures different fractions of glycated hemoglobin [1-3]. Most of the used methods are based on the chemical separation of the fractions of unglycated and glycated hemoglobin and subsequent determination of the concentration of glycated hemoglobin by subtracting the unbound form from the total amount of hemoglobin. These methods are invasive and have significant drawbacks, for example, chromatography is very sensitive to changes in temperature and pH [2].
In this study, the optical parameters of plasma and hemoglobin solution obtained from the whole blood of rats with developed model alloxan diabetes were measured, such as refractive indices in the visible and infrared spectral regions at temperatures from room to 50°C and fluorescence spectra for long-wave excitation 260, 270 and 280 nm.
For example, the results of measuring the optical properties of a hemoglobin solution obtained from rat whole blood by refractometric and fluorescent methods are presented. Hemoglobin samples were obtained by hemolysis of whole blood of rats with alloxan diabetes (Gl = 18.3 ± 2.2 g/l) and blood of healthy animals (Gl = 4.2 ± 0.8 g/l). The hemoglobin concentration in refractometric measurements was 40 g/l. Refractometric measurements were performed on an Abbe multiwavelength refractometer (Atago, Japan). The refractive index (RI) was measured at wavelengths of 480, 486, 546, 589, 644, 680, 930, 1100, 1300, and 1550 nm. The temperature dependence of the RI in the first approximation can be determined by the formula:
n(T) = n0(T = 0) + T*^ (1).
Thus, Figure 1(a) shows the initial value of the RI of the hemoglobin solution obtained from whole blood of control and diabetic animals, while Figure 1(b) and Table 1 show the temperature increment of RI. Both parameters were calculated by formula (1).
Control group Diabet group
400 600 800 1000 1200 1400 1600
Wavelength, nm
(a)
2.5
2.0
T—
— b
O 1.5
1 O1.0
T— *
0-5 0.0
Control group
480 nm 680 nm
486 nm 930 nm
546 nm 1100 nm
589 nm 1300 nm
644 nm 1550 nm
656 nm
Diabet group
(b)
Figure 1: The RI at 27 °C (A) and temperature increment (B) of RI of hemoglobin obtained by hemolysis of whole blood of healthy rats and rats with alloxan diabetes.
Table 1: The temperature increment of RI of hemoglobin obtained by hemolysis of whole blood of healthy rats and rats with alloxan diabete.
^Wavelength, nm
Group 480 486 546 589 644 656 680 930 1100 1300 1550
Control
group —— (*10"4 oC"1) 1.526 1.445 1.652 1.582 1.927 1.763 1.667 1.975 1.976 2.337 1.846
Diabetes group (*10"4 oC"1) 1.444 1.454 1.475 1.496 1.455 1.439 1.469 1.393 1.401 0.898 1.214
Control/Diabetes 1.057 0.994 1.120 1.057 1.324 1.225 1.135 1.418 1.410 2.601 1.520
Fluorescence spectra were measured using a Spectral Fluorolog-3 modular fluorimeter (HORIBA, Japan) and a Cary Eclipse fluorescent spectrophotometer (Varian, Belgium). The excitation wavelength was 260, 270, and 280 nm. The concentration for measuring fluorescence was chosen so that the absorption at the excitation wavelength did not exceed 0.1. (pH=6.35±0.14). The results of fluorescence measurements are shown in Figures 2(a), 2(b) and 2(c), the main parameters of the fluorescence spectra for the diabetic and control groups of animals are shown in Table 2.
Control group Diabet group
350 400 450 Wavelength,nm
50 ■ 40 -30 ■ 20 10 -0 -
Control group Diabet group
(a)
350 400 450 500 Wavelength, nm
(b)
3 50
é 40
I 30
u
300 350 400 450 Wavelength, nm
(C)
500
Figure 2: Fluorescence spectra of hemoglobin obtained by hemolysis of whole blood of healthy rats and rats with alloxan diabetes for excitation wavelength : A - 260 nm, B - 270 nm, C - 280 nm.
Table 2. Fluorescence Spectrum Parameters of hemoglobin obtained by hemolysis of whole blood of healthy rats and rats with alloxan diabetes for excitation wavelength : A - 260 nm, B - 270 nm, C - 280 nm.
Excitation wavelength Parameter Control group Diabetes group
260 nm ^max 344 339
FWHM 77 67
270 nm ^max 338 337
FWHM 87 71
280 nm ^max 340 338
FWHM 82 70
Analysis of the refractive indices and fluorescence spectra obtained from blood hemoglobin samples showed that the main differences between the control group of rats and animals with hyperglycemia were observed in relation to the temperature increment of the refractive index and fluorescence upon excitation of 260 nm.
Also for the dispersion dependences of RI hemoglobin and plasma, coefficients were calculated for the approximation of data according to the Sellmeier formula in the wavelength range of 480-1550 nm. For the temperature dependence of RI, normalization was carried out according to the maximum value of RI and the temperature increment of the normalized RI was calculated, for which a correlation with the content of free glucose was established. The correlation with the content of free glucose was established for the parameters of fluorescence spectra of hemoglobin and plasma obtained from whole blood of rats with developed model alloxan diabetes. A comparison was made between the results for animals with developed model alloxan diabetes and the results for a healthy group of animals in which the measured free glucose content in the blood was normal. The obtained results allow one to separate the healthy group of animals from the group with developed alloxan diabetes. The obtained data allow us to supplement the information available in the literature about changes in the optical characteristics of blood resulting from the development of a disease such as diabetes.
The reported study was funded by RFBR, project number 20-32-90058 and a grant under the Decree of the Government of the Russian Federation No. 220 of 09 April 2010 (Agreement No. 075-15-2021-615 of 04 June 2021).
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