CHARACTERIZATION OF PH VARIATION IN LYSED BLOOD BY NEAR-INFRARED SPECTROSCOPY

Near-infrared spectra (1300-2500 nm) collected from lysed blood soluti ons were shown to correlate,vith the pH of the solutions measured pote ntiometrically, Cross-validated partial least-squares (PLS) models wer e developed from these spectral data, which provided standard error of prediction (SEP) values below 0.05 pH units for a pH range of 1.0 (6. 8-7.8). Experiments were designed to eliminate possible correlation be tween pH and other components in the blood in order to ensure that var iations in the spectral data correlated to pH were due to hydrogen ion changes only. Further work was performed to discern the primary sourc e of pH information in the lysed blood spectra by using spectra collec ted from plasma and histidine solutions, The blood, plasma, and histid ine data sets were compared with the use of loading vectors from princ ipal component analysis (PCA). These loading vectors show that variati ons in the spectra of the titrated amino acid histidine mimic those se en in lysed blood, but not those seen in plasma. These results suggest that histidine residues of hemoglobin are providing the spectral vari ation necessary for pH modeling in the lysed blood solutions. It is fu rther shown that the observed pa-sensitive histidine bands do not aris e from the exchangeable proton on the imidazole ring of histidine; rat her they arise from the variation in the C-H bonds of the C2 and/or th e C4 carbons of the imidazole ring as they are influenced by the titra tion of the nitrogen-bound proton of the imidazole ring.