Resonance Raman in Vitro Detection and Differentiation of the Nitrite-Induced Hemoglobin Adducts in Functional Human Red Blood Cells

Abstract

This work presents in vitro studies of the functional, isolated human red blood cells (RBCs) treated with various concentrations of Na¹⁴NO₂ and Na¹⁵NO₂ with the use of resonance Raman spectroscopy (RRS) at two different laser excitations supported by absorption spectrophotometry (UV-vis). The products of the reaction between oxyhemoglobin (oxyHb) in isolated RBCs with NaNO₂ were analyzed and identified in situ. The metHb-H₂O was found to be the major product of this reaction; however, additional adducts were also clearly observed. Vibrational analysis allowed identification of various Hb³⁺NO₂ species (Fe³⁺-O-N=O with O-binding mode of nitrite ion to the Fe³⁺ core and nitrovinyl adducts with 2-vinyl nitration favored over 4-vinyl nitration) as well as the Fe³⁺-NO adduct. In addition, we were able to visualize in situ the Hb-NO₂ species inside functional RBCs with the use of Raman imaging.