In depth investigation of collagen non-enzymatic glycation by Raman spectroscopy.

Non-enzymatic glycation is a post-translational modification of long-lived matrix proteins such as type I collagen. It occurs during aging and leads to the formation of advanced glycation end-products (AGEs). AGE accumulation is associated with severe complications in chronic and age-related diseases. The assessment of modifications induced by this (patho)physiological process represents an interest in biology and medicine for a better patient care. The objective of our work was to position the interest of Raman spectroscopy in the quantification of collagen glycation. Two types of in vitro glycation were used by incubating collagen samples, at different durations, with ribose or glyoxylic acid; these reducing agents acting on the chemical specificity of the glycation reaction. Glycation efficiency was evaluated by the liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) quantification of carboxymethyllysine (CML) and pentosidine, which are among the most studied AGEs. Raman data were processed by PCA coupled to validity indices and Lasso regression as multivariate analysis tools. Regression models were constructed by considering the LC-MS/MS results as reference values. A marked variability was observed within the Raman datasets making difficult the identification of spectral differences between control and ribose-treated collagen samples. By taking advantage of the chemical specificity of the glyoxylic acid treatment leading to CML formation, on one hand, and the feature selection included in the Lasso algorithm, on the other hand, Raman markers associated with glycation were identified. The assigned vibrations corresponded to modifications of side chains of collagen. In addition, a threshold of CML concentration was determined as quantitative indicator of the applicability of Raman spectroscopy for potential patient follow-up purposes. Although lacking in sensitivity to directly detect AGEs in collagen, Raman spectroscopy allows to highlight the molecular modifications of collagen induced by glycation.