DOI 10.1515/cclm-2013-0174 Clin Chem Lab Med 2014; 52(1): 21–32 Review Vincent M. Monnier*, Wanjie Sun, David R. Sell, Xingjun Fan, Ina Nemet and Saul Genuth Glucosepane: a poorly understood advanced glycation end product of growing importance for diabetes and its complications Abstract: Advanced glycation end products (AGEs) repre- David R. Sell, Xingjun Fan and Ina Nemet: Department of Pathology, sent a family of protein, peptide, amino acid, nucleic acid Case Western Reserve University School of Medicine, Cleveland, OH, USA and lipid adducts formed by the reaction of carbonyl com- Saul Genuth: Department of Medicine, Case Western Reserve pounds derived directly or indirectly from glucose, ascor- University School of Medicine, Cleveland, OH, USA bic acid and other metabolites such as methylglyoxal. AGE formation in diabetes is of growing importance for their role as markers and potential culprits of diabetic com- plications, in particular retinopathy, nephropathy and neuropathy. Development of sensitive and specific assays Introduction utilizing liquid chromatography mass spectrometry with More than 35 years have elapsed since the landmark dis- isotope dilution method has made it possible to detect and covery of the clinical significance of hemoglobin A [1], quantitate non-UV active AGEs such as carboxymethyl- 1c a discovery that has unequivocally helped establish the lysine and glucosepane, the most prevalent AGE and pro- causal relationship between hyperglycemia, the patho- tein crosslink of the extracellular matrix. Below we review genesis of microvascular disease and the accelerated studies on AGE formation in two skin biopsies obtained progression of selected cardiovascular disease endpoints near the closeout of the Diabetes Control and Complica- in both type 1 and type 2 diabetes [2–6]. Concomitant tions Trial (DCCT), one of which was processed in 2011 for research during these years has successfully implicated assay of novel AGEs. The results of these analyses show glucose as a pathogenetic factor for diabetic retinopathy, that while several AGEs are associated and predict com- nephropathy, neuropathy and a co-factor in cardiovas- plication progression, the glucose/fructose-lysine/glu- cular disease in various animal models. In a nutshell, it is cosepane AGE axis is one of the most robust markers for widely accepted that diabetic complications are the result microvascular disease, especially retinopathy, in spite of of an intricate combination of oxidative and carbonyl adjustment for past or future average glycemia. Yet over- stresses and inflammatory signals that can be pharmaco- all little biological and clinical information is available on logically efficiently prevented in the experimental animal glucosepane, making this review a call for data in a field [7]. Paradoxically however, except for sustained lower- of growing importance for diabetes and chronic metabolic ing of glycemia over many years (DCCT, UKPDS), few to diseases of aging. none of the prevailing pharmacological anti-complica- tion paradigms [aldose reductase activation, oxidative Keywords: carbonyl stress; glycemia; HbA ; nephropathy; 1c stress, advanced glycation end products (AGE) formation, neuropathy; retinopathy; methyglyoxal. protein kinase C (PKC) activation, inflammation] have so far convincingly succeeded delaying complications in the *Corresponding author: Vincent M. Monnier, Department of human [7]. Pathology, Case Western Reserve University, Cleveland, OH 44106, Multiple reasons can be invoked as a reason why a USA, Phone: +1 216 3686613, Fax: +1 216 3681357, given drug was found unable to normalize the oxidant E-mail: [email protected] and carbonyl stress or their biological consequences in Vincent M. Monnier: Department of Biochemistry, Case Western diabetic humans. These may include poor bioavailability, Reserve University School of Medicine, Cleveland, OH, USA Wanjie Sun: Biostatistics Center, George Washington University, poor therapeutic window and thus inability to achieve Rockville, MD, USA; and Case Western Reserve University School of the necessary therapeutic dose, insufficient complica- Medicine, Cleveland, OH, USA tion event rate linked to overall improved management 22 Monnier et al.: Advanced glycation end product of growing importance for diabetes and its complications of diabetes, yet unrecognized pathogenetic mechanisms Glucosepane: chemistry and and genetic factors, or simply the inability to carry out the drug trial long enough until efficacy is demonstra- mechanism of formation ble. The latter proposition is supported by observations Glucosepane was discovered and so named by Lederer and which show that euglycemia for 5 years was insufficient colleagues, who isolated it from a model reaction of N-α- for improvement of morphological lesions of diabetic t-boc-L-lysine and N-α-t-boc-L-arginine and D-glucose at nephropathy and, instead, 10 years of euglycemia were 37°C [9]. The compound has a molecular weight of 647 Da needed [8]. and constitutes a mixture of four diastereoisomers with a Below, we develop arguments in support of the hypothesis that glycation damage to the extracellular 7-membered ring made of glucose that crosslinks the two matrix by glucose itself, and in particular glucosepane amino acid side chains of lysine and arginine (Figure 1). crosslinks, are among the factors that may explain the Glucosepane is labile to conventional acid hydrolysis and progression and slow reversibility of diabetic complica- therefore can only be quantitated in glucose exposed pro- tions in the human. The latter emphasis is based on the teins upon exhaustive mild enzymatic digestion at 37°C belief that diabetic rodents, while adequate models of to release the free molecule. The fact that glucosepane λ = hyperglycemic cellular stress are not adequate for probing absorbs UV light only at short wavelengths ( max 251 and the intrinsic role of the glucose-damaged extracellular 253 nm) makes it inconvenient for assay by HPLC using matrix (ECM) in diabetic complications, because of the absorption spectroscopy, and therefore the method of duration of the glycemic stress is too short for quasi irrep- choice is liquid chromatography mass spectrometry with arable damage to occur. isotope dilution technique (LC/MS) [9]. The first part of this review explores the molecular At first, the mechanism of glucosepane formation properties of glucosepane, its in vitro and in vivo mech- appears to be rather straight forward, proceeding via the anism of formation and inhibition, its distribution in key intermediate, N6-(2,3-dihydroxy-5,6-dioxohexyl)-L- animal and human tissue and how diabetes affects its lysinate (Lederer’s glucosone [10]) that can be trapped with levels. The second focuses on the recently uncovered clin- ortho-phenylenediamine (OPD) [11]. We recently proposed ical relationship between glucosepane, other skin AGEs that this intermediate is common to both glucose-derived and the presence of diabetic complications, the results of crosslinks, i.e., crossline and glucosepane (Figure 1). which lead us to propose a key role for glucosepane in the Crosslines are isomeric lysine-lysine crosslinks that have pathogenesis of diabetic complications. been described several years ago and immunochemically HN H R H N R 1 H N N R R 2 N 2 1 H N N+ 2 N O HO HO Arginine HO HO HN R1 HN R1 Glucosepane H2O O OH HO HO OH OH OH OH OH Amadori O HO OH O O OH N+ HO OH not found Amadori compound N6-(2,3-dihydroxy-5,6-dioxohexyl)- compund OH in vivo! -L-lysinate (Lederer's glucosone) H R 1 + N+ R1 N R1 Crossline NH NH R = 1 (CH2)4 R2= (CH ) Peptide backbone C 2 3 C O O Figure 1 Mechanism of formation of glucosepane from N6-(2,3-dihydroxy-5,6-dioxohexyl)-L-lysinate precursor. During in vitro incubation with free fructosyl-lysine in phosphate buffer significant amount of crossline are formed that are not detected under physiological conditions with proteins. Monnier et al.: Advanced glycation end product of growing importance for diabetes and its complications 23 detected in glucose exposed proteins and in vivo [12, 13]. this question and the potential cross-reactivity of “non- However, our investigations using mass spectrometry CML” antibodies cross-reacted with native glucosepane failed to detect these in protein digest from biological will need to be clarified. tissue, making it highly unlikely that crosslines are physi- ological crosslinks [14]. Similarly to the recently described AGE glucold, crosslines represent a class of glucose- derived AGEs that can form only in vitro under very high Glucosepane in biological systems: glucose concentrations from free but not protein-bound amino acids. effects of diabetes on tissue levels One important point to emphasize about glucosepane and protein turnover rate is that it forms entirely non-oxidatively from glycated lysine residues (Amadori products) via a series of water Relatively little information is yet available on tissue elimination reactions. Thus, this form of carbonyl stress levels of glucosepane (Table 1) and sites of formation in would proceed anaerobically in diabetes and be insen- proteins. In the collagen-rich extracellular matrix, glu- sitive to antioxidants! Interestingly, Nasiri et al. found, cosepane increases with age [18]. In normal human skin based on theoretical considerations, that glucosepane levels increase curvilinearly to reach about 2000 pmol/mg might possibly originate from a combination of methylgly- collagen at 100 years. However, in purified glomerular oxal and glyceraldehyde [15].