Probing non-enzymatic glycation of type I collagen: A novel approach using Raman and infrared biophotonic methods Marie Guilbert a , Georges Said a , Teddy Happillon a , Valérie Untereiner a , Roselyne Garnotel a , Pierre Jeannesson a , Ganesh D. Sockalingum a, ⁎ a Equipe MéDIAN-Biophotonique et Technologies pour la Santé, Université de Reims Champagne-Ardenne, Unité MEDyC FRE CNRS/URCA 3481, UFR de Pharmacie, 51 rue Cognacq-Jay, 51096 Reims, France abstract article info Article history: Received 9 October 2012 Received in revised form 8 January 2013 Accepted 13 January 2013 Available online 1 February 2013 Keywords: Type I collagen Glycation Advanced Glycation End products Fourier-transform infrared microspectroscopy Raman microspectroscopy Background: Non-enzymatic glycation is the main post-translational modification of long-life proteins observed during aging and physiopathological processes such as diabetes and atherosclerosis. Type I collagen, the major component in matrices and tissues, represents a key target of this spontaneous reaction which leads to changes in collagen biomechanical properties and by this way to tissue damages. Methods: The current study was performed on in vitro glycated type I collagens using vibrational microspectroscopies, FT-IR and Raman, to highlight spectral features related to glycation effect. Results and conclusions: We report a conservation of the triple-helical structure of type I collagen and notice- able variations in the exposure of proline upon glycation. Our data also show that the carbohydrate band can be a good spectroscopic marker of the glycation level, correlating well with the fluorescent AGEs formation with sugar addition. General significance: These non-invasive and label-free methods can shed new light on the spectral features of glycated collagens and represent an effective tool to study changes in the extracellular matrix observed in vivo during aging or on the advent of a pathological situation. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Type I collagen is the most abundant extracellular matrix protein in the human body. It is assembled in triple-helical structure, and cross-linking between triple helices allows it to form a fibrillar network [1]. Because of its long lifespan which can be variable, about 15-years in skin, collagen I undergoes post-translational modi- fications during aging or pathological conditions such as diabetes mellitus [2] and more recently in cancer processes [3,4]. The main modification is a non-enzymatic glycation [5,6], resulting in the fixation of reducing agents, such as aldose sugars, essentially on lysine residues of type I collagen. In the field of cancer, scaffolds of glycated collagens have been used to study the impact of this post-translational modifica- tion on tumor cell proliferation [7] and migration [8]. Further, in tissue engineering strategies, glycated collagen has also been shown to be of great interest for preparing cartilage constructs [9,10], mimicking diabetic wound healing [11] or evaluating the effect of mechanical constraints on cellular behavior [12]. At the mechanistic level, glycation leads to the formation of Schiff bases that are transformed into Amadori products. After complex rearrangements, the Amadori compounds give rise to irreversible products called advanced glycation end products (AGEs) [3]. Among these end products, N E -carboxymethyllysine (CML) and pyrraline have been found to be non-fluorescent and non-cross-linking, while pentosidine and crossline have been described as fluorescent and cross-linking compounds [13–15]. AGEs contribute to changes in the collagen properties such as loss of the triple helix solubility and flex- ibility, resulting in an increase of its rigidity [1]. Consequently, via these structural and molecular modifications of collagen triple helix, its enzymatic digestivity is less efficient with aging and in pathologi- cal situations like diabetes [16]. Glucose is the major blood circulating sugar in the human body but it exhibits less reducing properties than agents such as ribose, glyceraldehydes or fructose [4,17]. Although these latter compounds are less involved in in vivo protein glycation processes, they are able to generate elevated AGEs levels and are therefore used for in vitro collagen glycation [1]. Protein glycation evaluation is currently assessed by conventional spectrometric, chromatographic or immunohistochemical methods. To detect post-translational modifications of the collagen and to provide a quantitative determination of resulting cross-links, mass spectrometry and high performance liquid chromatography have Biochimica et Biophysica Acta 1830 (2013) 3525–3531 ⁎ Corresponding author at: FRE CNRS/URCA no. 3481, UFR Pharmacie, 51 rue Cognacq-Jay, 51096 Reims Cedex, France. Tel.: +33 3 26 91 35 53; fax: +33 3 26 91 35 50. E-mail addresses: marie.guilbert@univ-reims.fr (M. Guilbert), georgessaid@gmail.com (G. Said), teddy.happillon@gmail.com (T. Happillon), valerie.untereiner@univ-reims.fr (V. Untereiner), roselyne.garnotel@univ-reims.fr (R. Garnotel), pierre.jeannesson@univ-reims.fr (P. Jeannesson), ganesh.sockalingum@univ-reims.fr (G.D. Sockalingum). 0304-4165/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagen.2013.01.016 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen