Oliver Reihl

Cross‐Linking of the Extracellular Matrix by the Maillard Reaction in Aging and Diabetes: An Update on “a Puzzle Nearing Resolution”

Abstract: The aging extracellular matrix is characterized by an age‐related increase in insolubilization, yellowing, and stiffening, all of which can be mimicked by the Maillard reaction in vitro. These phenomena are accelerated in metabolic diseases such as diabetes and end‐stage renal disease, which have in common with physiological aging the accumulation of various glycation products and cross‐links. Eight years ago we concluded that the evidence favored oxidative cross‐linking in experimental diabetes [Monnier, V.M. et al. 1996. The mechanism of collagen cross‐linking in diabetes: a puzzle nearing completion. Diabetes 45(Suppl. 3): 67‐72] and proposed a major role for a putative non‐UV active cross‐link derived from glucose. Below, we provide an update of the field that leads to the conclusion that, while oxidation might be important for Maillard reaction‐mediated cross‐linking via Strecker degradation and allysine formation, the single most important collagen cross‐link known to date in diabetes and aging is glucosepane, a lysyl‐arginine cross‐link that forms under nonoxidative conditions.

Formation Pathways for Lysine-Arginine Cross-links Derived from Hexoses and Pentoses by Maillard Processes UNRAVELING THE STRUCTURE OF A PENTOSIDINE PRECURSOR

Covalently cross-linked proteins are among the major modifications caused by the advanced Maillard reaction. So far, the chemical nature of these aggregates and their formation pathways are largely unknown. Synthesis and unequivocal structural characterization are reported for the lysine-arginine cross-linksN 6-{2-{[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino}-5-[(2S,3R)-2,3,4- trihydroxybutyl]-3,5-dihydro-4H-imidazol-4-ylidene}-l-lysinate (DOGDIC 12),N 6-{2-{[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino}-5-[(2S)-2,3-dihydroxypropyl]-3,5-dihydro-4H-imidazol-4-ylidene}-l-lysinate (DOPDIC 13), and 6-((6S)-2-{[(4S)-4-ammonio-5-oxido-5-oxopentyl] amino}-6-hydroxy-5,6,7,7a-tetrahydro-4H-imidazo[4,5-b] pyridin-4-yl)-l-norleucinate (pentosinane 10). For these compounds, as well as for glucosepane 9 and pentosidine 11, the formation pathways could be established by starting from native carbohydrates, Amadori products, and 3-deoxyosones, respectively. Pentosinane 10 was unequivocally proven to be an important precursor of pentosidine 11, which is a well established fluorescent indicator for advanced glycation processesin vivo. The Amadori products are shown to be the pivots in the formation of the various cross-links 9–13. The bicyclic structures 9–11 are directly derived from aminoketoses, whereas 12 and 13 stem from reaction with the 3-deoxyosones. All products 9–13 were identified and quantified from incubations of bovine serum albumin with the respective 3-deoxyosone or carbohydrate. From these results it seems fully justified to expect both glucosepane 9 and DOGDIC 12 to constitute important in vivo cross-links.

Identification and quantitative evaluation of the lysine-arginine crosslinks GODIC, MODIC, DODIC, and glucosepan in foods

The presence of the various protein crosslinks GOLD 2, MOLD 3, GODIC 4, MODIC 5, DODIC 6, and glucosepan 7 in foods has been established for the first time by liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization (ESI). In compounds 2 and 3 two lysine moieties, in 4-7 a lysine and an arginine side chain are joined by the crosslink. Unequivocal identification of 2-7 was achieved with independently synthesized reference material. The quantitative results for the investigated foodstuffs show MODIC 5 to be the most important Maillard crosslink. The concentrations of 5 and GODIC 4 are 5-10 fold higher than those of the corresponding imidazolium compounds 3 and 2, establishing 5 and 4 as the major food protein crosslinks derived from methylglyoxal and glyoxal, respectively. The maximum value of 151 mg MODIC 5/kg protein (equivalent to 0.42 mmol/kg protein) was found in a butter biscuit sample which also shows the highest overall Maillard crosslink content with 0.71 mmol 47/kg protein. These first quantitative results suggest that compounds 4-7 can be jointly responsible for protein polymerization in the course of food processing.

Formation pathways for lysine–arginine cross-links derived from hexoses and pentoses by Maillard processes

Abstract α-Dicarbonyl compounds are crucial intermediates in the cross-linking of proteins by reducing sugars in the course of the Maillard reaction. The novel dideoxyosones N6-(2,3-dihydroxy-5,6-dioxohexyl)lysine (9a,b) and N6-(2-hydroxy-4,5-dioxopentyl)lysine (12) were unequivocally identified via their triazine derivatives and these α-diketo compounds established as precursors of the major in vivo cross-links glucosepane 11 and pentosidine 16, respectively. This new dideoxyosone class is formed in high yield in the model incubations and the underlying reaction sequence will improve our understanding of protein cross-linking and of Maillard chemistry in general.

Spiro cross-links: Representatives of a new class of glycoxidation products

Covalently cross-linked proteins are among the major modifications caused by the advanced Maillard reaction. So far, the chemical nature of these aggregates is largely unknown. Investigations are reported on the isolation of 6-[2-[[(4S)-4-amino-4-carboxybutyl]amino]-6,7-dihydroxy-6,7-dihydroimidazo[4,5-b]azepin-4(5H)-yl]-L-norleucine (10) and N-acetyl-6-[(6R,7R)-2-[[4-(acetylamino)-4-carboxybutyl]amino]-6,7,8a-trihydroxy-6,7,8,8a-tetrahydroimidazo[4,5-b]azepin-4(5H)-yl]-L-norleucine (12) formed by oxidation of the major Maillard cross-link glucosepane 1. Independent synthesis and unequivocal structural characterization are given for 10 and 12. Spiro cross-links, representing a new class of glycoxidation products, were obtained by dehydrogenation of the amino imidazolinimine compounds N6-[2-[[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino]-5-[(2S,3R)-2,3,4-trihydroxybutyl]-3,5-dihydro-4H-imidazol-4-ylidene]-L-lysinate (DOGDIC 2) and N6-[2-[[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino]-5-[(2S)-2,3-dihydroxypropyl]-3,5-dihydro-4H-imidazol-4-ylidene]-L-lysinate (DOPDIC 3). These new oxidation products were synthesized, and their unambiguous structural elucidation proved the formation of the spiro imidazolimine structures N6-[(7R,8S)-2-[[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino]-8-hydroxy-7-(hydroxymethyl)-6-oxa-1,3-diazaspiro[4.4]non-1-en-4-ylidene]-L-lysinate (16), N6-(8R,9S)-2-[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino]-8,9-dihydroxy-6-oxa-1,3-diazaspiro[4.5]dec-1-en-4-ylidene)-L-lysinate (19), and N6-[(8S)-2-[(4-amino-4-carboxybutyl)amino]-8-hydroxy-6-oxa-1,3-diazaspiro[4.4]non-1-en-4-ylidene]-L-lysinate (18), respectively. It was shown that reaction of the imidazolinone 15 led to the formation of spiro imidazolones, structurally analogous to 16 and 19.