Reaction, or non-enzymatic browning. Glucosepane is found to form through a non-oxidative path. The exact mechanism leading to glucosepane has been a challenge for researchers to determine. However, it is currently well characterized up to the ring formation. The formation of glucosepane within connective tissues has been shown to be site-specific. For example, studies using Molecular Dynamics simulations of a complete collagen fibril revealed energetically favourable locations, particularly within the collagen fibril gap-region. This may be due to the lower protein density and higher intra-fibrillar water content within the gap-region. Overall reaction pathway The overall pathway of glucosepane formation starts with lysine attacking the reducing sugar D-glucose to form the unstable imine known as a Schiff base, which then rearranges to form the more stable aminoketose Amadori product. From there, the stable Amadori Product slowly degrades to form glucosepane through an α-dicarbonyl intermediate. Mechanism of α-dicarbonyl formation from the Amadori product The particular reaction path proceeding from the Amadori product to the α-dicarbonyl intermediate that will yield glucosepane was difficult to determine. Initially, researchers hypothesized an α-dicarbonyl intermediate in which the carbonyls were located on C-2 and C-3 of D-Glucose. However, by using glucose with C-1, the carbonyl carbon, marked with the isotope 13C in the reaction, researchers found that the α-dicarbonyl formed has the carbonyls located at C-5 and C-6 of the original glucose backbone. The best mechanism proposed is that the α-dicarbonyl N 6-(2,3-dihydroxy-5,6-dioxohexyl)-L-lysinate, a key intermediate in the glucosepane reaction, forms from the Amadori product through a carbonyl shift all the way down the 6 carbon sugar backbone by keto-enol tautomerism and the elimination of the C-4 hydroxyl. Further, evidence was given for the extent of the hypothesized carbonyl shift by using heavy hydrogen in the solvent water, D2O. Researchers found that all the H-C-OH of the carbon backbone were converted to D-C-OH after the reaction, demonstrating that all the hydrogens got transferred out through keto-enol tautomerism, and thus the carbonyl shift went all the way down the backbone, finally eliminating the C-4 hydroxy group. Ring closure to arginine cross-linking It is still relatively unclear how the ring is formed and when. One article suggests, and it seems the current belief, that the ring must form in the step after the α-dicarbonyl is formed. The study hypothesized, and another found correlating evidence, that the most likely mechanism of getting from the α-dicarbonyl to glucosepane is through the