Assessment of carboxyhemoglobin content in the blood with high accuracy: wavelength range optimization for nonlinear curve fitting of optical spectra

Abstract

The impact of carbon monoxide (CO) gas on the human organism is very dangerous. The affinity of CO to hemoglobin is considerably higher than that of oxygen. Thus, the interaction of CO with the blood results in a higher content of carboxyhemoglobin (HbCO) in red blood cells (RBCs) and correspondingly in tissue hypoxia. The disruption in the organism depends on the HbCO content in the blood. To assess any complications in the body at a given moment due to CO exposure and predict future consequences, it is necessary to measure the dynamics of hemoglobin derivative concentrations simultaneously. However, measuring HbCO and other derivatives in RBCs without hemolysis accurately is complicated due to the strong intercollinearity between the molar absorptivities of hemoglobin derivatives and superposition of absorption and scattering spectra. In the present study, to quantitatively assess the contents of the hemoglobin derivatives in the blood after exposure to CO, improved accuracy is achieved by optimizing the wavelength range used for the nonlinear curve fitting of optical spectra. Experimental spectra were measured in the wavelength range $\Delta \lambda =500-700\text{nm}$ . For each experimental curve, it was established the value of optimal interval $\Delta {\lambda }_{opt}$ for which the correlation coefficient between experimental data and corresponding points of the theoretical fitting curve was the maximum in the wavelength range $\Delta {\lambda }_{typ}=535-580nm$ , which contains the typical absorption peaks for $Hb{O}_2$ , $Hb$ , and $HbCO$ . The concentrations obtained based on such fitting curves were considered to be highly accurate. The quantitative assessment enabled the determination of $\text{the}HbCO$ nonlinear increase with the time of CO exposure in the in vitro experiment and the study of the dynamics of hemoglobin derivative transformations during blood incubation.

Keywords: Absorption and scattering; Biochemistry; Biophysics; Carbon monoxide; Carboxyhemoglobin; Cell biology; Hematological system; Intensive care; Intercolinearity of molar absorptivities; Mathematical biosciences; Medical physics; Nonlinear curve fitting; Physics methods; Spectrophotometry; Toxicology.

Figure 1

Experimental stages. 1 - preparation of the blood suspension for the control and CO exposure samples; 2 – the dilution of stage (1) suspension for the measurement of optical spectra; 3 - measurement of the absorption spectra by spectrophotometry; 4 – analysis of optical spectra; 5 – the system for the CO exposure in blood.

Figure 2

Optical spectra of the RBC suspensions: (a) Wavelength range $\Delta \lambda =500-700nm$; (b) Wavelength range $\Delta \lambda =530-585nm$ (detailed image). 1 - $t_{exp}=0\text{s }\left(\text{control}\right)$, 2 - $t_{exp}=80\text{s}$, 3 - $t_{exp}=160\text{s}$, 4 - $t_{exp}=320\text{s}$. Incubation time$t_{inc}=1\text{hour}$.

Figure 3

Choosing the wavelength interval for the accurate quantitative assessment of the content of hemoglobin derivatives in the blood by nonlinear curve fitting of optical spectra (control blood): (a) $\Delta {\lambda }_A=$ 500–700 nm; (b) $\Delta {\lambda }_B=$ 500–650 nm; (a) $\Delta {\lambda }_C=$ 500–614 nm; (d)–(f) detailed graphs at $\Delta {\lambda }_{typ}=535-580nm$ corresponding to (a)–(c), the correlation coefficients $\text{τ}{\text{ }}_{exper/theor\left(fitting\right)}$ and the sum of squared deviations ${\delta }^2$ are indicated for (d)–(f). 1- experimental data, 2 – fitting curve.

Figure 4

Wavelength intervals $\Delta {\lambda }_{opt}$ for the accurate quantitative assessment of the contents of the hemoglobin derivatives in the blood by the nonlinear curve fitting (2) of experimental optical spectra (1): (a) $\Delta {\lambda }_{opt}=500-590nm$, t_exp = 0 s (control), t_inc = 1 h, C(HbCO) = 0.8%; (b) $\Delta {\lambda }_{opt}=500-590nm$, t_exp = 80 s, t_inc = 1 h, C(HbCO) = 52.8%; (c) $\Delta {\lambda }_{opt}=510-590nm$, t_exp = 320 s, t_inc = 1 h, C(HbCO) = 87.9%; (d) $\Delta {\lambda }_{opt}=520-660nm$, t_exp = 320 s, t_inc = 24 h, C(HbCO) = 41.6%.

Figure 5

Dynamics of hemoglobin derivative transformation: (a) the change of $C\left(Hb{O}_2\right)+C\left(Hb\right)$ (1), $C\left(MetHb\right)\left(2\right)$, $C\left(HbCO\right)$ (3) for different CO exposure times t_exp; (b) photograph of RBC suspensions – control and after CO exposure; (c) experimental data for the increase of $C\left(HbCO\right)$ (3) with t_exp and the corresponding theoretical fitting curve; (d) experimental data for decreasing $C\left(Hb{O}_2\right)+C\left(Hb\right)$(1) with t_exp and the corresponding theoretical fitting curve.

Figure 6

Experimental optical spectra and diagrams of the calculated concentrations of $C\left(Hb{O}_2\right)+C\left(Hb\right)$(1), $C\left(MetHb\right)\left(2\right)$, $C\left(HbCO\right)$ (3) for different incubation times t_inc= 1 h and t_inc= 24 h: (a), (d) t_exp = 0 s (control); (b), (d) for t_exp = 320 s.