Atomic force microscopy and Raman spectra profile of blood components associated with exposure to cigarette smoking

Abstract

Tobacco smoke contains several compounds with oxidant and pro-oxidant properties with the capability of producing structural changes in biomolecules, as well as cell damage. This work aimed to describe and analyse the effect of tobacco smoke on human blood components, red blood cell (RBC) membrane, haemoglobin (Hb) and blood plasma by Atomic Force Microscopy (AFM) and Raman spectroscopy. Our results indicate that tobacco induced RBC membrane nano-alterations characterized by diminished RBC diameter and increased nano-vesicles formation, and RBC fragility. The Raman spectra profile suggests modifications in chemical composition specifically found in peaks 1135 cm^-1, 1156 cm^-1, 1452 cm^-1 and intensity relation of peaks 1195 cm^-1 and 1210 cm^-1 of blood plasma and by change of peaks 1338 cm^-1, 1357 cm^-1, 1549 cm^-1 and 1605 cm^-1 associated with the pyrrole ring of Hb. The relevance of these results lies in the identification of a profile of structural and chemical alterations that serves as a biomarker of physiological and pathological conditions in the human blood components induced by tobacco exposure using AFM and the Raman spectroscopy as tools for monitoring them.

Fig. 1. RBC membrane by AFM. (a) Nano-structure of RBC membrane of non-smokers and smokers volunteers and mean square roughness (mean ± SD), n = at least 15 cells per group. (b) Profile line showing membrane depressions.

Fig. 2. RBC osmotic fragility. The percentage of haemolysis increases significantly (p < 0.001) in the RBC of smokers 5–9 and smokers >10 in comparison with RBC of non-smoker participants.

Fig. 3. Average of Raman spectra of blood plasma. Black line represents the average of non-smokers (n = 6, no. of spectra 100), red line represents the average of smokers <5 (n = 4, no. of spectra 40). Blue line represents the average of smokers 5–9 (n = 4, no. of spectra 40) and magenta line represents the average smokers >10 (n = 3, no. of spectra 30). Only relevant peaks from analysis are marked. Intensity relation between peak 1210/1195 is showed in figure amplification. Asterisks indicate significant differences compared with non-smokers (p < 0.05).

Fig. 4. Score plot PC1 vs. PC2 for blood plasma. Ellipses are confidant area. Black circles are non-smokers; red squares are smokers <5; blue triangles are smokers 5–9; magenta inverted triangles are smokers >10.

Fig. 5. Analysis of Raman spectroscopy of erythrocytes. Black line represents the average of non-smokers participants (n = 120). Red line represents the average of smokers <5 (n = 80), blue line represents the average of smokers 5–9 (n = 80). The magenta line represents the average of smokers >10 (n = 60).

Fig. 6. Score plot PC1 vs. PC2 for haemoglobin. Ellipses are confidant area. Black circles are non-smokers controls; red squares are smokers <5; blue triangles are smokers 5–9; magenta inverted triangles are smokers >10.