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Historical Development

Louis Camille Maillard (1878 – 1936) is celebrated by this Society as the first scientist to investigate the “browning reaction”. He investigated the reaction between glucose and glycine on heating [1]. He proposed that the brown pigmented product or melanoids involved an initial interaction between amines and saccharides forming Schiff’s base adducts. This is now called the “Maillard reaction”. Other scientists were also interested in similar reactions in food and physiological chemistry. In 1908, Robert Ling produced flavoured and coloured compounds from heat drying proteins with sugars such as glucose and maltose between 120°C to 150°C. He called these compounds glucosamine-like products [2]. These were amongst the earliest recorded adducts formed between monosaccharides and amines.

In 1898, Pinkus observed the formation of methylglyoxal-bis-phenylhydrazone on incubating glucose in dilute sodium hydroxide with phenylhydrazine [3]. He concluded that similar reactions may occur in physiological systems. Pinkus was one of the first investigators, therefore, to observe the degradation of glucose to a reactive α-oxoaldehyde, methylglyoxal. The glyoxalase system that catalyses the conversion of methylglyoxal to lactate was discovered in 1912 [4;5] – the example of an enzymatic defence against glycation. For the next 30 years, methylglyoxal was investigated as an intermediate of glycolytic metabolism. With the discovery of a series of phosphorylated saccharides derivatives in the metabolism of glucose in mammalian tissues by Embden, Meyerhof and co-workers [6;7], methylglyoxal was discarded as a intermediate of mainstream glycolysis. In keeping with this view, the glyoxalase system was later found to metabolise methylglyoxal to D-lactate and not the L-lactate of mammalian glycolysis [8].

In 1925 -31, Mario Amadori demonstrated that the condensation of D-glucose with aromatic amines p-phenetidine, p-anisidine or p-toluidine gave two structurally different isomers which were not anomers [9;10]. One of the isomers was more labile than the other towards hydrolysis and was also more susceptible to decomposition on standing in the solid state in air. He correctly recognised this as the N-glycosylamine but he mistakenly thought that the more stable isomer was a Schiff’s base, overlooking its resistance to acid hydrolysis [10;11]. In 1936, Kuhn and Dansi found that the stable isomer was not a Schiff’s base but rather the product of a molecular rearrangement. They also confirmed that the labile isomer was in fact the N-substituted glycosylamine [12]. In 1937, Kuhn and Weygand reported the structure of the Amadori’s stable isomer, which was the unbranched N-substituted 1-amino-1-deoxy-2-ketose [13]. Kuhn and Weygand later called the reaction the Amadori rearrangement involving generally aldoses and amines. In 1953, Hodge suggested that the Maillard reaction involved formation of a Schiff’s base followed by Amadori rearrangement in the early stages. He also suggested that fructosamine degraded to glucosone and other adducts via enolisation, oxidation and fragmentation reactions [14].

In 1960, Anet reported the degradation of a fructosamine (N,N-difructosylglycine) to 3-deoxyglucosone [15]. This was the first example of the importance of fructosamine dehydration to a 3-deoxyglucosone (3-DG). Kato also isolated 3-deoxyglucosone and 3-deoxypentosone from the browning reactions of glucose and ribose with an amine [16]. These reactive α-oxoaldehyde products of the Maillard reaction are now seen as important precursors of glycation adduct formation in biological systems.

In 1965, Albert Szent-Gyorgyi proposed methylglyoxal and other α-oxoaldehydes s cell growth retarding substances or “retine” and the glyoxalase system which detoxified α-oxoaldehydes a cell growth promoting substance or “promine” [17]. Later, it was found that growth factors, hormones and other factors were the critical growth controlling substances and the retine-promine theory of Szent-Gyorgyi was discarded.

In 1968, Samuel Rahbar [18] reported a fast-moving hemoglobin band in cellulose acetate electrophoresis that was particularly evident in blood samples of diabetic patients. This was the discovery of the glucose derived Amadori products of hemoglobin, glycated hemoglobin HbA1c – now widely used to assess glycemic control in diabetic patients.

In 1973, Bonsignore and co-workers presented the first evidence that a triosephosphate, glyceraldehyde-3-phosphate, degraded non-enzymatically under physiological conditions to form methylglyoxal. This reaction could now explain why Neuberg and co-workers many years earlier [19] found methylglyoxal formation in mammalian tissues incubated with fructose-1,6-bisphosphate – which is converted to triosephosphates by aldolase.

In 1975, Koenig produced conclusive evidence of the formation of Amadori products in haemoglobin using 1H-NMR spectroscopy [20]. In the mid-1970s, measurement of the concentration of HbA1c was developed as an indicator of glycaemic control over the proceeding 6 - 8 weeks in diabetic patients [21-24].

In 1977, Takahashi described the reaction of amino acids with glyoxal derivatives, including glyoxal and methylglyoxal. Arginine was identified as the predominant amino acid modified and a hydroimidazolone was one molecular structure proposed for the adducts – although not supporting analytical data were given [25;26;26].

In 1980, Kato et al. reported the formation of 6-(2-formyl-5-hydroxymethylpyrrol-1-yl)-L-norleucine from 3-deoxyglucosone and lysyl residues in proteins, now commonly known as pyrraline, which was an advanced glycation endproducts (AGE) of the Maillard reaction between D-glucose and L-lysine [27]. Hayashi and Namiki presented evidence for the fragmentation of the saccharide moiety early in the Maillard reaction, leading to the formation of α-oxoaldehydes [28]. Evidence for the formation of glyoxal and methylglyoxal (presented later) established saccharide fragmentation as a new series of reaction pathways in Maillard chemistry, now collectively called the Namiki pathway.

In 1984, the first structure of the product formed from the physiological degradation of Amadori product, 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI) was reported by Cerami et al. and was thought to be the recognition factor for the uptake of advanced glycated and aged proteins [29]. This compound is now considered of limited physiological relevance and was formed during sample processing [30-32]. Also, Wolff and co-workers reported the slow oxidative degradation of monosaccharides under physiological conditions to form the corresponding α-oxoaldehyde and hydrogen peroxide. This process was called monosaccharide autoxidation [33].

In 1986, Cerami first used the term advanced glycation endproducts (AGE) to refer to “brown fluorescent pigments which crosslink proteins” formed from the degradation of fructosamine [34]. Cerami and co-workers observed increased collagen cross-linking in the arterial walls of diabetic rats and associated fluorescence characteristic of AGE compounds. The nucleophilic hydrazine derivative, aminoguanidine (PimagedineTM), inhibited the formation of AGEs and diabetes-induced protein crosslinking [35;36]. Baynes and co-workers also reported the formation of Nε-carboxymethyl-lysine (CML) and erythronic acid from the degradation of glycated proteins, and also noticed that these two compounds were present in human urinary metabolite [37]. Monnier and co-workers also detected the presence of the pyrraline in serum albumin of human subjects, which was increased significantly in diabetic subjects [38].

In 1988, Thornalley presented evidence linking hyperglycemia in diabetes mellitus with increased flux of formation and concentration of methylglyoxal [39]. Baynes and co-workers reported the formation of the Nε-lactatolysine and the co-product glycerate by oxidative degradation of fructosamine residues of glycated proteins. Nε-lactatolysine was present in human lens collagen and excreted in urine [40].

In 1989, Monnier and Sell isolated an acid stable fluorescent compound from collagen – “pentosidine”. It was a crosslink formed from a pentose moiety with lysine and arginine residues [41]. This has become one of the most widely studied AGEs.

In 1990, Szwergold and co-workers identified the presence of a 3-phosphokinase activity in lenses of rats that converted fructose to fructose-3-phosphate. The later metabolite spontaneously degraded to 3-DG [42]. In 1991, the enzymatic metabolism of fructosamine adducts was also discovered. Tatsuo Horiuchi and co-workers identified fructosyl-amino acid oxidase in extracts from a soil-derived bacteria. The enzyme catalysed the conversion of fructosyl-amino acids to the free amino acid, glucosone and hydrogen peroxide [43]. These studies indicate that there are enzymatic mechanisms that convert fructosamine and fructosamine to 3-DG.

In 1992, Stern and co-workers identified an AGE receptor protein from bovine lung endothelial cells – later shown to have a sequence mass of 42 kDa [44]. This was the Receptor for Advanced Glycosylation Endproducts (RAGE) and is the best characterised of the AGE receptors. RAGE binds AGE-modified proteins and other ligands - β-amyloid protein and amphoterin. RAGE is now implicated in vascular dysfunction associated with diabetic complications, macrovascular disease and uraemia, and also neurite outgrowth, Alzheimer’s disease and metastasis of tumours. The truncated extracellular domain of RAGE, soluble RAGE, was found to block RAGE-mediated cell activation by AGE proteins and is now under development for therapeutic applications. RAGE related glycation research is one of the most promising applications of molecular biological techniques to glycation-linked physiological processes.

In 1993, the glycation of the basic phospholipid PE was studied by Bucala and co-workers – a first demonstration of the glycation of phospholipids [45]. In 1994, Henle and co-workers [46] identified the presence of methylglyoxal-derived hydroimidazolone in protein of food stuff (pretzel crust). Thornalley and co-workers showed that proteins glycated by methylglyoxal and by glucose contained significant modifications of arginine residues as well as the expected lysine modification. Proteins glycated by glucose and by methylglyoxal were bound and internalised by cell surface receptors [47;48].

In 1995, Vaca and co-workers used a 32P-post-nuclease digest labelling techniques for trace nucleotide analysis and showed the presence of guanyl nucleotides glycated by methylglyoxal in human lymphocyte DNA. The adduct formed was an imidazopurinone derivative [49]. Studies by Bucula and co-workers showed that methylglyoxal-derived adducts were formed from the modification of DNA by fructosamine and an additional nucleotide adduct, N2-(1-carboxyethyl)guanine was identified [50]. The glycation of DNA has been associated with mutagenesis and activation of nucleotide excision repair of DNA.

In 1996, Cerami and co-workers investigated a potential pharmacological strategy for selectively cleaving the glucose-derived protein crosslinks, they found that N-phenacylthiazolium bromide (PTB) reacted with and cleaved covalent, AGE-derived protein crosslinks. The ability of PTB to break AGE crosslinks in vivo offered a potential therapeutic approach for the removal of established AGE crosslinks [51]. Later studies showed other effects were involved; PTB rapidly hydrolysed during pharmacological evaluation, acidifying the test systems and generating a thiol-containing hydrolysis products which reduced protein disulphides [52].

Over the period 1994-1998, the inhibitor of AGE formation aminoguanidine (PimagedineTM) was in clinical trial for the prevention of overt nephropathy in type 1 diabetic subjects (ACTION I) and type 2 diabetic subjects (ACTION II). In 1998, ACTION II was discontinued because of lack of efficacy and safety concerns. In ACTION I, aminoguanidine failed to achieve the clinical endpoint – decrease of the time to the doubling of baseline serum creatinine. Aminoguanidine is unlikely to find clinical use due to lack of efficacy and toxicity – reviewed in [53].

In 2000, Takashi Tsuruo and colleagues discovered overexpression of glyoxalase 1 produced multidrug resistance in human tumours [54]. This revealed that nucleotide glycation by glyoxalase 1 substrates, glyoxal and methylglyoxal, may be involved in the mechanisms of induction of apoptosis of clinical antitumour agents and glycation research has a critical role in countering multidrug resistance in clinical drug therapy.

In 2003, quantitative screening of glycation, oxidation and nitration biomarkers of proteins was introduced for the first time using LC-MS/MS. This provided the first quantitative and comprehensive screening of protein glycation adducts. High levels of glycation adducts in cellular protein, free glycation adducts in plasma and urine, and the marked (up to 50-fold) accumulation of glycation free adducts in uraemia were found for the first time [55]. The first conference was held on the enzymatic defence against glycation: it was also recognised for the first time that there are a group of enzymes involved in the suppression of glycation processes and repair of glycated proteins in physiological systems. Enzymes involved are: glyoxalase I, aldehyde reductases and dehydrogenases, Amadoriase and fructosamine-3-phosphokinase [56].

Currently, glycation processes are thought to contribute to disease processes – diabetic complications, macrovascular disease, Alzheimer’s disease, cirrhosis, uraemia, arthritis, ageing and others. The measurement of glycation adducts is a critical clinical, diagnostic tool in assessment of glycaemic control in diabetes mellitus. The prevention or repair of glycation adducts is a strategy for the development of novel therapeutic agents under investigation in many research laboratories. In many cases, glycation adducts are risk markers of disease but whether glycation adducts are risk factors of disease is still unknown. There remains continuing debate about the physiological significance of AGEs in food. It is clear that food is a rich source of AGEs but highly glycated proteins may not be digested efficiently, and AGEs that are absorbed are probably mainly glycated amino acids or AGE free adducts which are excreted rapidly in the urine by subjects with normal renal function. Glycation research remains an area of outstanding interest, fascination and innovation in chemistry, biology, medicine, food, nutrition and technological and applied sciences.


Recommended reading:


Kawamura S, 70 Years of the Maillard-Reaction. ACS Symposium Series 215: 3-18, 1983.

Rahbar S, The discovery of glycated hemoglobin: a major event in the study of nonenzymatic chemistry in biological systems. Ann NY Acad Sci 1043: 9-19, 2005.


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