An In Vitro Study on the Non-Enzymatic Glycation of Melamine and Serum Albumin by Reducing Sugars

Glycation is a non-enzymatic reaction with reactants including a reducing sugar and a free amino containing molecule such as protein, amino acids, DNA, RNA and lipids. In the initial phase of glycation, the carbonyl group of the reducing carbohydrate condenses with the free amino groups on the target biomolecule to form reversible glycosylamines, which are then converted to more stable Amadori products. Once formed, these Amadori products can with time undergo dehydration, cyclization, oxidation, and rearrangement to form a polymorphic group of compounds collectively referred to as Advanced Glycation Endproducts (AGEs). The accumulation of AGEs in vivo has been implicated as a major pathogenic process in diabetic complications including diabetic cataract formation, retinopathy and neurological diseases, as well as other health disorders, such as Alzheimer’s disease (AD). Chapter one reviewed the chemistry of glycation and the formation of AGEs in a mechanical perspective. The role of AGEs in the pathogenesis of diabetic nephropathy, diabetic neuropathy, diabetic retinopathy and Alzheimer’s disease were considered. Here we also reviewed the potential inhibitors against glycation published to date, focusing on some novel potential AGE inhibitors such as zinc and gold nanoparticles. The purpose of the study described in chapter two was to investigate the susceptibility of the amine groups of melamine to glycation by milk sugars and sugar metabolites. Dairy products adulterated with melamine have been recently blamed for the death of at least several infants and the sickening of countless children in China. The presented study described the non-enzymatic glycation of melamine with milk sugar D-galactose and several sugar metabolites including methylglyoxal, glyoxal and DL-glyceraldehyde. The chemical structures of melamine AGEs were characterized by electrospray mass spectrometry. The factors influencing the rate and extent of melamine’s glycation were also evaluated. The third part of the dissertation described a study on anti-glycation effect of gold nanoparticles (GNPs). In this study we showed that certain sizes of spherical GNPs exhibit an inhibitory effect on the formation of AGEs when Bovine Serum Albumin (BSA) was glycated by D-ribose. A combination of UV spectrometry, HPLC and circular dichroism showed that only GNPs with size ranging from 2nm to 20nm inhibited the formation of BSA AGEs. The inhibition effect of GNPs was correlated to the overall surface area of nanoparticles in the solution. GNPs with higher surface areas were found to be better inhibitors of glycation, whereas those with low surface areas were less effective inhibitors. The inhibitory effect of GNPs on non-enzymatic glycation reactions may be due to the covalent bonding between gold atoms on the surface of GNP and ε amino groups of L-lysine residue on protein. In chapter four, we evaluated the effect of UVC radiation on glycation of Human Serum Albumin (HSA). In this study, we found that exposure to UVC radiation accelerated the glycation level of HSA and promoted the formation of AGEs, which may be stimulated by the generation of ROS by UVC radiation. A combination of several analytical methods including UV spectrometry, HPLC, and MALDI-TOF were used to evaluate the glycation level of HSA at 37 °C under neutral pH in the presence of D-glucose in vitro. This study warrants further investigation as there have been few reports on the correlation between the UVC radiation of proteins and their enhanced glycation by a reducing sugar.

sugars and sugar metabolites. Dairy products adulterated with melamine have been recently blamed for the death of at least several infants and the sickening of countless children in China. The presented study described the non-enzymatic glycation of melamine with milk sugar D-galactose and several sugar metabolites including methylglyoxal, glyoxal and DL-glyceraldehyde. The chemical structures of melamine AGEs were characterized by electrospray mass spectrometry. The factors influencing the rate and extent of melamine's glycation were also evaluated.
The third part of the dissertation described a study on anti-glycation effect of gold nanoparticles (GNPs). In this study we showed that certain sizes of spherical GNPs exhibit an inhibitory effect on the formation of AGEs when Bovine Serum Albumin (BSA) was glycated by D-ribose. A combination of UV spectrometry, HPLC and circular dichroism showed that only GNPs with size ranging from 2nm to 20nm inhibited the formation of BSA AGEs. The inhibition effect of GNPs was correlated to the overall surface area of nanoparticles in the solution. GNPs with higher surface areas were found to be better inhibitors of glycation, whereas those with low surface areas were less effective inhibitors. The inhibitory effect of GNPs on non-enzymatic glycation reactions may be due to the covalent bonding between gold atoms on the surface of GNP and ε amino groups of L-lysine residue on protein.
In chapter four, we evaluated the effect of UVC radiation on glycation of Human Serum Albumin (HSA). In this study, we found that exposure to UVC radiation accelerated the glycation level of HSA and promoted the formation of AGEs, which may be stimulated by the generation of ROS by UVC radiation. A combination of several analytical methods including UV spectrometry, HPLC, and MALDI-TOF were used to evaluate the glycation level of HSA at 37 °C under neutral pH in the presence of D-glucose in vitro.
This study warrants further investigation as there have been few reports on the correlation between the UVC radiation of proteins and their enhanced glycation by a reducing sugar.

Introduction
The Non-enzymatic reaction between the carbonyl group of a reducing sugar and the amino containing molecule such as protein, peptide or amino acid was first studied under defined conditions by Louis Camille Maillard in 1912. Ever since its description in early 1900s, the Maillard reaction has continued to be a topic of research interest. For researchers in nutrition science, harnessing the Maillard reaction has allowed for controlling food flavor, food aroma, food coloring, and food texture. In addition to its benefits, the Maillard reaction occurring during food processing also results in the loss of protein quality and the formation of harmful compounds with mutagenic, carcinogenic and genotoxic properties (1-3).
During the 1970s and 1980s, researchers in clinical medicine realized that this process also occurs slowly in vivo. Subsequently, Maillard reaction in vivo was termed glycation, distinguished from enzymatic glycosylation.
The final products of this reaction are a polymorphic group of compounds collectively referred to as Advanced Glycation Endproducts (AGEs). The gradual built-up of AGEs in body tissues plays an important role in the pathogenesis of diabetic complications including atherosclerosis, renal failure and cardiovascular disease. Glycation is also found to be related to other health disorders such as Alzheimer Diseases (AD) and normal aging (4)(5)(6).
Intracellular hyperglycemia can induce the formation of glucose-derived dicarbonyl molecules such as glyoxal, methylglyoxal and 3-deoxyglucosone.
These intracellular dicarbonyls, also called AGE precursors, are much more reactive comparing to D-glucose due to their lack of cyclic structure and comparatively smaller size. AGE precursors glycate with amino groups of both intracellular and extracellular proteins at a much faster rate than D-glucose, leading the accumulation of AGEs. These AGEs specifically binds to a certain groups of cell surface receptors (RAGEs), inducing receptor-mediated production of reactive oxygen species (ROS), finally resulting in oxidative cellular dysfunction (3,7). AGEs such as glyoxal lysine dimer (GOLD) and methylglyoxal lysine dimer (MOLD) are responsible for protein crosslinking on longest-lived extracellular proteins such as collagen and elastin. Such proteins provide strength and flexibility to tissues, and do not get recycle very often. Glycation and crosslinking gradually change the property and function of these proteins, reduce their flexibility and elasticity, and increase their stiffness as well. This process has been implicated as strong contributors to many progressive diseases of aging, including vascular diseases, stiffness of joints, arthritis and kidney failure (4-6).
Both synthetic compounds and natural products have been found to be inhibitors against the formation of AGEs. These glycation inhibitors are proposed to block the formation of intermediate Amadori products, or break the crosslinking of proteins. Their classification and corresponding inhibition mechanism will be discussed later in this article.

The chemistry of glycation
Glycation is a non-enzymatic browning reaction that involves a series of steps with the reactants typically including a reducing sugar and a free amino containing molecule. The progress of the reaction includes three distinguishable phases: the initial stage, the intermediate stage and the late stage. In the initial phase of glycation, the carbonyl group of a reducing carbohydrate condenses with the free amino group on a protein or other amino containing molecule to form reversible glycosylamine, which is then converted to more stable Amadori products. Under appropriate condition, these Amadori products could degrade to more reactive dicarbonyls such as methylglyoxal, glyoxal and glyceraldehyde. These intermediates are responsible for most of the AGE formation due to their high reactivity. In the late stage of glycation, the Amadori product itself could with time undergo dehydration, cyclization, oxidation, and rearrangement to form AGEs.
Meanwhile, the AGE precursors formed from the intermediate stage of glycation such as methylglyoxal will in turn interact with protein or other amino containing molecules to produce AGEs at a much higher reaction rate comparing to reducing sugars.

Initial stage: formation of Amadori products
In the initial phase of glycation, the nitrogen atom of a terminal -amino group or of the ε-amino group on lysine or arginine residue covalently condenses with the carbonyl group of a reducing sugar by nucleophilic attack. The product of this reaction contains an unstable aldimine structure named Schiff base. The formation of Schiff base is reversible due to the unstable property of aldimine functional group. Schiff base intermediate can then undergo Amadori rearrangement to produce a relatively stable ketosamine structure, referred to as Amadori product (See figure 1).
Formation of Amadori product from Schiff base is much faster than the reversed reaction so Amadori adducts tend to accumulation on proteins.
This process is thought to be facilitated by localized acid-base catalysis if there is a histidine or lysine side chain about 5 Å from the target amino residue (8).

Intermediate stage: formation of AGE precursors
AGE precursors are formed in vivo via two major pathways: 1) the fragmentation of Amadori products or Schiff base, and 2) the metabolic degradation of D-glucose. The physiologically relevant D-glucose is one of the least reactive reducing sugars in protein glycation (9). Under physiological condition, over 90% of D-glucose is in cyclic confirmation which cannot be a target of the nucleophilic attack by a primary amino group.
However, other sugars and dicarbonyl compounds, many of which are found intracellular such as glyoxal (Gly), 3-deoxyglucosone (3-DG) and methylglyoxal (MG) participate in glycation at a proportionally faster rate (10,11). Gly is formed from several reactions such as the oxidative fragmentation of Schiff base via the Namiki pathway (12,13), glucose auto-oxidation catalyzed by metal (Wolff pathway) or lipid peroxidation via the Acetol pathway (14,15). 3-DG is produced from non-oxidative fragmentation and hydrolysis of Amadori product. 3-DG also forms from fructose-3-phosphate, an intermediate of the Polyol pathway in which glucose is reduced to sorbitol by aldose reductase. Further degradation of 3-DG generates methylglyoxal and glyceraldehyde (GA) by retro-aldol condensation (16,17). Methylglyoxal is found to be one of the most reactive glycation agents, and could react with different amino acids including lysine and arginine to generate dicarbonyl-derived AGEs. MG could also alter the secondary structure of a protein, resulting in the loss of its function (18,19).
A summary of the sources of these reactive carbonyl species are shown in figure 2.
The increase in the concentration of reactive carbonyl compounds from glycoxidation, lipoxidation or the degradation of Schiff base/Amadori adducts will lead to carbonyl stress (20,21), a situation that aggravates the modification of proteins and prompts the formation of AGEs. Figure 3 is a list of chemical structures of currently well-defined AGE dicarbonyl precursors and their interactions with glycated proteins.

Late stage: formation of AGEs
AGEs are a group of heterogeneous compounds including fluorescent crosslinking structures such as pentosidine, non-fluorescent crosslinking substances such as glyoxal lysine dimer (GOLD) or methylglyoxal lysine dimer (MOLD), and non-fluorescent, non-crosslinking compounds such as N ε -carboxymethyl lysine (CML). AGEs are produced both in vitro and in vivo via several pathways, including direct degradation of Amadori products or Schiff base, protein modification by dicarbonyl compounds and reactions between Amadori products and AGE precursors.
One of the major consequences of protein being modified by active dicarbonyl glycation agents is the formation of protein cross-links, a situation that is related to several diabetic complications. Reactions of dicarbonyl compounds with lysine and arginine residue or with Amadori intermediate are involved in the formation of many AGE crosslinking structures which exhibit fluorescence absorbance ( Figure 4). Among these compounds, pentosidine from the glycation of ribose has been determined in many tissues such as lens proteins and skin collagens (22). Pentosidine can also be formed from several more reactive carbohydrate intermediates including 3-DG and fructose. It has been recently proposed as an index for evaluating tissue damage under glycation condition (23).
Although fluorescent crosslinking AGEs are important biomarkers of hyperglycemia condition due to their easy of detection, these structures are account for only a small portion of protein crosslinks which is about 1% in physiological condition (24). On the other hand, non-fluorescent crosslinking AGEs are the major contribution to in vivo protein-protein crosslinks. A group of most well defined crosslinking structures are imidazolium crosslinks including glyoxal-derived AGE glyoxal-lysine dimer (GOLD) and methylglyoxal-derived AGE methylglyoxal-lysine dimer (MOLD) (25,26).
Both of these structures are found in human lens protein and human serum. (27)(28)(29). Glyoxal and methylglyoxal could irreversibly modify the ε-amino group of lysine residues in protein under physiological condition to form these imidazolium compounds which are responsible for protein crosslinking.
The third class of AGEs is non-fluorescent, non-crosslinking structures, which act as biological receptor ligands to initiate cellular signaling and induce tissue oxidation stress. These compounds could also perform as precursor of protein crosslinks. The chemical structures of some of the most important and well-studied non-crosslinking AGEs including pyrraline, 1-carboxyalkyl lysine and imidazolone A and B are shown in figure 5.
3-DG rapidly reacts with lysine residues of a protein to form pyrraline.
Though pyrraline itself is not a crosslink structure, it may cause crosslinks between proteins when its aldehyde group forms a Schiff base with another amino group, resulting in lysine-lysine or lysine-arginine crosslinking in vivo.
Imidazolone is derived from reaction of dicarbonyl compounds with the guanidino group of an arginine residue.
AGEs such as N ε -carboxymethyl lysine (CML) and N ε -carboxymethyl lysine (CEL) with a carboxyalkyl group attached to the ε-amino group of an amino acid are found in vivo. CML is known as one of the most well characterized AGEs which is used as a biomarker for long-term protein damage in diabetes and oxidative tissue abnormality. CML can be produced through different pathways including glycation and oxidation reactions (22,30,31), resulting in its wide distribution in a variety of tissues such as serum, skin collagen and kidney.
CML is produced through four major pathways shown in figure 6: 1) oxidative cleavage of Amadori product 2) Schiff base degradation through Namiki pathway (32); 3) oxidation of ascorbic acid (33) and 4) autoxidation glycosylation with lysine and glyoxal (32). The oxidative degradation of Amadori adducts is thought as the major source of CML formation (25,34).
Under physiological condition, CML is found to be produced mainly from fructoselysine, an Amadori intermediate derived from fructose and lysine.
The Schiff base can undergo degradation via Namiki pathway to form active carbonyl compounds such as glyoxal and its corresponding imine analog, which are responsible for the formation of CML. The other CML precursor formed via Namiki pathway is aminoaldehyde. Aminoaldehyde could oxidize to CML by any of the oxygen reactive species present in the glycation system, or by oxygen under metal catalysis. Other sources of CML formation include the oxidation of ascorbic acid derived from glucose and direct condensation of glyoxal with the ε-amino group of lysine residues in proteins.

Biological effects of glycation
Non-enzymatic glycation has been thought to play an important role in the pathogenesis of diabetic complications including cataract formation, renal failure and cardiovascular disease. Glycation is also approved to be related to other health disorders such as Alzheimer's disease (AD), atherosclerosis and normal aging. Most of the effects of glycation which lead to chronic complications are due to AGE accumulation on long-lived proteins such as collagen and lens crystallins. In the meantime, glycation can induce the formation of reactive oxidation species (ROS), causing oxidation stress and tissue damage (35, 36).

Physiological defense system against glycation
There are several defense systems occurring physiologically against the deleterious effects of AGE, including enzymes such as aldose reductase， which inhibit the formation of AGE precursors or antioxidants such as glutathione which reduce the production of reactive oxidative species.
Aldose reductase is a NADPH-dependent enzyme that catalyzes the degradation of dicarbonyl AGE intermediates including MG and 3-DG (37-39). Most recently, fructoselysine, an Amadori intermediate derived from fructose and lysine, was found to be phosphorylated by human fructosamine-3-kinase (FN3K) and spontaneously degradation afterwards (40). The glyoxalase system catalyzes the conversion of methylglyoxal to D-lactic acid by glyoxalase I and glyoxalase II. This system is present in the cytosol and mitochondria of cells and has a wide distribution in living organisms (41, 42). Another natural common defense mechanism is glutathione (GSH) system. GSH is an antioxidant, preventing oxidative damage to tissues caused by reactive oxidative species such as peroxides and free radicals, whose formations are prompted by glycation. GSH also facilitated the rearrangement of methylglyoxal to D-lactate in the glyoxalase pathway (43).

AGE mediated cell-signaling
Intracellular accumulation of AGEs damages target cells through receptor-mediated pathway, in which the glycated plasma proteins bind to AGE receptors on the cell surface, inducing cell signaling and oxidation stress (see figure 7). Several AGE binding proteins have been identified such as RAGE, the AGE receptor complex (AGE-RG) and macrophage scavenger receptor (44-46), many of which play a critical part in the pathology associated with AGE related complications. In endothelial cell system, plasma proteins modified by AGEs act as ligands to AGE receptors found on the surface of the cell. This process activates intracellular transducers and leads to the changes in gene expression. A rise in the expression of pro-inflammatory and pro-coagulatory molecules was found in endothelial cell when it was exposed to high glucose (47-50). RAGE is a member of the immunoglobulin receptor family. Pro-inflammatory NF-κB pathway is thought to be one of the major targets of RAGE. The activation of the pathway is related to several inflammatory diseases in diabetes. In addition, NF-κB in turn elevates RAGE expression, establishing a positive feed-back cycle. In summary, the intracellular accumulation of AGE results in the increase of RAGE ligands, activating NF-κB pathway and lead to chronic inflammation.
Additionally, AGE receptors are found on the surface of macrophage and mesangial cell. Binding of AGE ligands to the receptor induces the formation of reactive oxygen species (ROS) including peroxyl, superoxide anions, hydroxyl radicals and hydrogen peroxides (structures seen in figure   8). As discussed previously in this article, the abnormal excess of ROS under hyperglycemia conditions originate from several mechanisms including the degradation of glycation intermediates, the by-production of carbonyl AGE precursors, and cellular oxidant stress triggered by RAGE.
The abnormal accumulation of ROS caused by hyperglycemia is known to mediate the progression of several chronic complications due to the impaired antioxidant defense system. Such situation is defined as oxidative stress. Pathways for the generation of ROS which lead to oxidative stress are summarized in figure 9. The rise in the oxidant level in a living organism can cause damage to various cell components and triggers the activation of certain cell signaling pathways, resulting in pathological changes in gene expression.

AGE induced protein dysfunction
Several intracellular proteins modified by glycation are found to have altered activities and stabilities. One example is serum albumin, the most abundant protein in blood plasma which exhibits a broad range of physiological functions such as catalytic activities. Serum albumin can catalyze the hydrolysis of esters, showing an esterase-like activity. However, its enzymatic activity is impaired drastically when the protein is glycated by methylglyoxal. Since a lysine residue of the protein has been suggested as the catalytic center, the loss of the protein's esterase activity is thought to be related to the modification of this lysine residue by glycation agents (51).
Another example of dysfunctional AGE modified protein is proteases, a class of enzymes which degrade polypeptides. The catalyze sites of this enzymes include cysteine and histidine, both of which can be glycated by methylglyoxal, resulting in the loss of enzymes' activity (52).
AGE formation does not only alter the activity of intracellular proteins, but affect the functional properties of important molecules on extracellular matrix such as various types of collagens. Collagen is the main components of connective tissue, responsible for skin elasticity, blood vessel strength and tissue regeneration. This type of proteins has a comparatively long biological half-life and is exposed to extracellular glucose whose level is considerably higher than that inside the cell. Collagen can react with other collagen molecules through protein crosslinking by some typical AGE structures including methylglyoxal lysine dimer (MOLD) and glyoxal lysine dimer (GOLD). Such crosslinking structures can be gradually built up during normal aging and are accumulated much faster under hyperglycemia condition. As a result, the formation of MOLD and GOLD plays a critical role in the pathologenesis of vascular stiffening and atherosclerotic lesions (17).

AGEs and diseases
The accumulation of AGEs has been implicated as strong contributors to many progressive diseases including diabetic complications, aging and Moreover, the balance of nitroso-redox can be disturbed by the formation of ROS when the radicals inactive relaxing nitric oxide and cause hypertension.
Besides the activation of PKC and the up-modulation of ROS, the accumulation of AGEs causes the increased synthesis of extracellular matrix and decreased expression of its degrading enzymes, leading to the polymerization and expansion of extracellular matrix proteins (56-58).

Diabetic neuropathy
The symptoms of diabetic neuropathy often develop slowly over years, including the loss of sensation, nausea, deep pain in legs and feet. The pathology can be characterized by progressive nerve fiber lose caused by long-term high blood sugar level in diabetes. The precise mechanisms underlying diabetic neuropathy is unclear, however most recent study suggests that the loss in nerve fiber can be caused by multiple pathways including the enhanced polyol pathway under hyperglycemia condition, the increased protein modification by AGEs and the up-regulated oxidation stress.
In polyol pathway, glucose is degraded to sorbitol by aldose reductase.
This process can be up-regulated under hyperglycemia condition, causing increased consumption of NADPH and reduced formation of glutathione.
The impaired synthesis of glutathione will then cause vascular insufficiency and degeneration of nerve fibers (59). The activation of polyol pathway also suppresses the activity of protein kinase C, and eventually elicits reduced nerve blood flow and decreased nerve conduction (60). Meanwhile, the binding of AGEs to their receptors has been found to induce oxidative stress, result in over expression of pro-inflammatory genes, and exaggerate neurological dysfunction, including altered pain sensation. The AGE-RAGE interaction can also activate the formation of extracellular matrix proteins.
AGEs in terns covalently bond to these proteins to form crosslink structures, and lead to the development of vascular abnormalities, such as thickening of capillary basement membrane.

Diabetic retinopathy
Many in vitro and in vivo studies showed that AGEs play a significant pathogenic part in diabetic retinopathy, when the retinal microvascular Besides diabetic retinopathy, the cataract formation is another major complication developing gradually in diabetes disorder. Under hyperglycemia condition, there is an increased production of highly reactive dicarbonyl compounds such as methylglyoxal and glyoxal. These molecules not only act as AGE precursors but also cause AGE crosslinks and protein aggregations on crystallins, leading to the formation of cataract (64)(65)(66).
Other study suggests that oxidation stress, which is upgraded by glycation, can promote the formation of cataract as well.

AGEs and Alzheimer's Diseases
Alzheimer's disease (AD) is an age-related chronic complication which severely affects patients' memory and behavior in their late life. The causes of such disease include early stage triggering events and late stage inflammation and neurodegeneration. Several studies suggest that the formation of AGEs plays an important role in the pathogenesis of AD.
The deposition of β-Amyloid (Aβ) in the cerebral cortex is one of the major pathological features of AD. Aβ peptides contain 39 to 43 amino acid residues and are found overproduced in AD brains. These peptides are thought to be one of the most important contributors to neurodegeneration (67,68). Solid evidence revealed that extensive protein crosslinking and the increased production of ROS, inflammatory factors and reactive dicarbonyl compounds are responsible for AD characteristics including the Aβ deposit (69). Meanwhile, all the above mentioned chemical processes are related to glycation and AGEs, whose formation is found to be accelerated in AD.
It has been suggested that the accumulation of AGEs is involved in the pathogenesis of AD through two major mechanisms including AGE-RAGE interaction and protein-Aβ crosslinking. As described in the previous chapter, AGE modified proteins can interact with cell surface receptors such as RAGE, and trigger a serial of cellular signaling pathways. Besides AGEs, Aβ is found to be another ligand of RAGE. The interactions of AGEs and Aβ with RAGE elicit the production of ROS and pro-inflammatory factors. The generation of these molecules will then cause oxidative stress and inflammatory, subsequently leading to neuronal dysfunction. The accumulation of AGEs is also responsible for protein-Aβ crosslinks, a major cause to neurodegeneration in AD. Such binding down-regulates the expression of genes involved in Aβ clearance, resulting in Aβ accumulation.

AGEs and aging
Hyperglycemia is shown to be correlated to several age-related

Inhibitors against AGE formation
Since non-enzymatic glycation of proteins is a major contributor to the pathology of diabetes and other diseases including AD, the inhibitors to prevent the formation of AGEs have been extensively investigated and a number of potential AGE inhibitors have been proposed over years. A schematic of in vivo glycation reactions is shown in figure 10. On this scheme are also common targets for AGE inhibition which are labeled by circled letters from A to F. Type A and type B inhibitors prevent the formation of Amadori product at the initial stage of glycation by competing with glycation agents (type A) or reacting with reducing sugars (type B) (70)(71)(72).
Other types of inhibitors (type C1 and C2) disturb glycation mechanisms such as Wolff pathway and Namiki pathway, enhance antioxidant activity, and reduce the production of dicarbonyl AGE precursors. Type D drugs interact with reactive dicarbonyl compounds such as methylglyoxal and glyoxal, preventing their binding to protein (73,74). Comparing to class D, class E compounds directly act on Amadori adducts, and trap it from further modification (75). Instead of preventing AGE formation, drugs in F group reduce AGE toxicity by breaking protein-AGE cross-linking. There are also numbers of novel AGE inhibitors such as zinc and nanoparticles, whose inhibition mechanisms are still under investigation.

Carbonyl compounds scavengers
The first and most well-known synthetic glycation inhibitor is aminoguanidine (AG) (76), whose structure can be found in figure 11.
Aminoguanidine prevents the formation of AGEs by reacting with reactive dicarbonyl compounds such as methylglyoxal and glyoxal, hindering the late stage of glycation. Several in vivo studies demonstrated that AG retarded the development of several diabetes related complications including renal failure and cataract formation (77). Unfortunately, AG showed considerable toxicity to human body during clinical trial, thus structural modification on the compound is required to reduce its toxicity. Pyridoxamine (PM), one of the vitamin B complexes, is another potent carbonyl scavenger which showed better inhibitory effect on reducing the glycation level in bovine serum albumin model (78). PM inhibits the formation of AGEs not only by trapping the reactive dicarbonyl AGE precursors, but by blocking the oxidative degradation of the Amadori intermediates as well, preventing it from further modification by reducing sugars (79).

AGE breakers
Cell tissue constantly exposed to AGEs tent to lose its function due to protein crosslinking. Recently, a few AGE breakers have been discovered to combat deleterious protein cross-links. Their structures are shown in figure   12. This class of anti-glycation agents provides a potential new therapeutic approach especially for diabetic cardiovascular disease. Patients who received alagebrium chloride (ALT-711), one of the most well studied AGE breakers, showed significant reduction in arterial pulse pressure (80).

Dietary antioxidant
A dietary antioxidant is considered to be relatively safe for human consumption comparing to above mentioned synthetic compounds. The accumulation of AGEs induces the formation of ROS, leading to cell dysfunction and tissue damage. Dietary antioxidants are substances found in food that decrease the damage of ROS, and restore physiological function in humans. Vitamin C and vitamin E, for instance, both prevented the formation of AGEs in experimental models. Treatment of diabetic rats with these antioxidants resulted in a reduced plasma lipid peroxidation level.
The study also showed that vitamin E has a potent to restore nerve system by reversing nerve conduction velocity deficits (82).

Novel AGE inhibitors
Besides synthetic anti-glycation agents and traditional dietary substances which reduce the damage of ROS, some novel AGE inhibitors have also been proposed recently, including zinc and gold nanoparticles (GNP). In a recent in vitro study, human serum albumin treated with different concentrations of zinc showed lower glycation level comparing to control experiment, suggesting that zinc inhibited the formation of AGEs. In this study, a combination of analytical methods was used to evaluate the level of protein glycation. Moreover, the treatment of zinc not only reduced the formation of glycation products, but also retained protein's secondary structure, an observation which was confirmed by circular dichroism. Zinc was proposed to inhibit glycation by binding to the reactive sites on the protein and preventing sugar's non-enzymatic condensation with these sites (83). GNP is another substance which showed anti-glycation activity as well as satisfying bio compatibility. In one study, spherical gold nanoparticles (GNPs) with a diameter of 2 nm showed an inhibition effect on the formation of AGEs when human serum albumin was glycated with DL-glyceraldehyde.
The inhibitory effect of GNPs on non-enzymatic glycation reactions may be due to the covalent bonding between gold atoms on the surface of GNP and ε amino groups of L-lysine residue on protein. As GNP is highly biocompatible and water soluble, an insight of its anti-glycation effect and its inhibition mechanism on the formation of AGEs might lead to a novel therapeutic application of GNP on reducing AGE related complications (84).

Conclusion
In conclusion, glycation is a non-enzymatic reaction with reactants including a reducing sugar and a free amino containing molecule such as protein, amino acids, DNA, RNA and lipids. In the initial phase of glycation, the carbonyl group of the reducing carbohydrate condenses with the free amino groups on the target biomolecule to form reversible glycosylamines, which are then converted to more stable Amadori products. Once formed, these Amadori products can with time undergo dehydration, cyclization, oxidation, and rearrangement to form a polymorphic group of compounds collectively referred to as Advanced Glycation Endproducts (AGEs). The accumulation of AGEs in vivo has been implicated as a major pathogenic process in diabetic complications including diabetic cataract formation, retinopathy and neurological diseases, as well as other health disorders, such as aging and Alzheimer's disease (AD).

Abstract
Melamine (1,3,5-triazine-2,4,6-triamine) is employed in the manufacture of plastics, laminates and glues, yet, it has been found sometimes added illegally to dairy products to artificially inflate foods' protein content. In 2008, dairy products adulterated with melamine were blamed for the death of several infants in China, a situation that forced Beijing to introduce stricter food safety measures. The objectives of this study were threefold: 1) to investigate, using UV and fluorescence spectrometry, the susceptibility of the amine groups of melamine to glycation with D-galactose, D-glucose and lactose, sugars commonly found in milk, 2) to study the rate and extent of melamine's glycation with methylglyoxal, glyoxal and DL-glyceraldehyde, three highly reactive metabolites of D-galactose, D-glucose and lactose, and 3) to characterize, using HPLC and mass spectrometry, the Advanced Glycation Endproducts (AGEs) of melamine with sugars found commonly in milk and their metabolites. Incubation of D-galactose, D-glucose and lactose with melamine revealed that D-galactose was the most potent glycator of melamine, followed by D-glucose, then lactose. Methylglyoxal, glyoxal, and DL-glyceraldehyde glycated melamine more extensively than D-galactose, with each yielding a broader range of AGEs. The nonenzymatic modification of melamine by sugars and sugar-like compounds warrants further investigation, as this process may influence melamine's toxicity in vivo.

Introduction
In 2008, many dairy products in China were found to be adulterated with the chemical melamine, a situation that contributed to the death and hospitalization of children and a massive recall of many food products (1 -5).
The adulteration of food products with melamine unfortunately continues to this day in China, despite the government's ongoing efforts to halt this practice.
Melamine is an organic base with a 1,3,5-triazine backbone. That is essentially a trimer of cyanamide with three amino groups attached to its hexagonal structure (see figure 1). The three amino groups suggest that melamine should be a target for nonenzymatic glycation by the Maillard reaction.
The Maillard reaction is a non-enzymatic browning reaction that involves a series of steps with the reactants typically including a reducing sugar and a protein. In the initial phase of the Maillard reaction, the carbonyl group of the reducing carbohydrate condenses with the free amino groups on the protein to form reversible glycosylamines, which are then converted to more stable Amadori products. Once formed, these Amadori products can with time undergo dehydration, cyclization, oxidation, and rearrangement to form a polymorphic group of compounds collectively referred to as Advanced Glycation Endproducts (AGEs) (6 -8).
Ever since its description in 1912, the Maillard reaction has continued to be a topic of research interest. One reason for this focus on the Maillard reaction is that it cuts across several disciplines with nutrition science and medicine serving as examples. For researchers in nutrition science, harnessing the Maillard reaction has allowed for control of food flavor, food aroma, food coloring, and food texture (9 -10). For researchers in clinical medicine exploring the Maillard reaction has meant gaining a deeper insight into the biochemistry of diabetes and its chronic complications including renal failure, cataract formation and atherosclerosis (11 -13).
Studies in our laboratory and those of others have shown that AGEs can form both in vitro and in vivo, and that besides proteins can involve other free amino containing molecules such as DNA, RNA, lipids, amino acids and amino sugars (14 -17). The objectives of this study were three fold: first, to determine if melamine can be glycated by by D-galactose, D-glucose and lactose, three sugars that are present in milk; second, to compare the glycation levels of melamine with D-galactose, D-glucose and lactose; third, to determine if melamine can nonenzymatically react with methylglyoxal, glyoxal and DL-glyceraldehyde, three highly reactive metabolic products of D-galactose, D-glucose and lactose (15; 18 -21).
In this report, we show that melamine is susceptible to glycation by D-galactose and demonstrate the ability of melamine to form AGEs. We also describe the nonenzymatic glycation of melamine with methylglyoxal, glyoxal, and DL-glyceraldehyde, the three more reactive metabolites of milk sugars. Emphasis is also placed on the characterization of the AGEs of melamine by HPLC, UV, fluorescence and mass spectrometry. were placed at -20 °C until analyzed.

UV and fluorescence spectroscopy
UV readings were obtained at a wavelength of 240 nm with an UltroSpec 2100 instrument (Biochrom Ltd, Cambridge, UK). Fluorescence measurements were made at respective excitation and emission wavelengths of 260 nm and 380 nm using a Spectra Max M2 spectrometer (Molecular Devices, Sunnyvale, CA). All readings were obtained in thermostatically controlled cuvettes that were maintained at 25±1 °C. The above excitation and emission wavelengths were determined optimal for detecting melamine AGEs.

High-performance liquid chromatography
Each HPLC run was performed in triplicate using a Hitachi system (San Jose, CA, USA) equipped with a low-pressure gradient pump (L-2130), a four-channel degasser, a sequential auto sampler (L-2200), and a high sensitivity diode-array detector (190-800 nm) (L-2455). AGE species were separated on a C 8 reverse phase HPLC column (5 μm×4.6 mm×150 mm) and monitored at 240 nm. Mobile phase consisted of a 92% solution of 10 mM citrate heptane sulfonate buffer, pH 3.0 and 8% acetonitrile. An isocratic condition was applied for 15 min at a constant flow rate of 1.00 ml min -1 . Prior to HPLC analysis, all solvents were filtered with a 0.45 μm membrane (Millipore, Billerica, MA, USA), degassed for 15 min, and centrifuged.

Mass spectrometry
Mass spectrometry studies were performed on a ThermoFinnigan LCQ mass spectrometer (ThermoScientific, Waltham, MA) equipped with an electrospray ionization source and a quadrupole ion trap mass analyzer.
Samples were diluted into a solvent consisting of 50/50 (v/v) 0.1% acetic acid in water/acetonitrile and directly infused into the electrospray source using a capillary syringe pump at a flow rate of 3 μL min -1 . Nitrogen was used as the sheath gas (setting at 60), and ultrapure helium was used as the collision gas. The ion spray voltage was set as 4.5 kV and the capillary temperature was 210 °C. The mass spectrometer was set to function in the positive ion mode with parameters optimized during direct infusion of caffeine standards with solvent. Samples were analyzed by MS and tandem mass spectrometry (CID fragmentation with Helium) favoring isolation and fragmentation of singly charged ions. The ESI/MS system was operated with the Xcalibur software (version 2.0, ThermoFinnigan), with the same software used also for data analysis. open-chain configuration [23,24]. Thus, with a higher percentage of the sugar existing in an open ring structure than D-glucose, D-galactose becomes more vulnerable to nucleophilic attack by the amino groups on melamine [23,24]. This phenomenon may also explain galactose's higher reactivity than D-lactose since as a disaccharide, lactose contains D-glucose at it reducing end. Increases in melamine concentration in the presence of set amounts of the various aldehydes also promoted AGE formation, demonstrating that the rate of glycation reaction was dependent on both the concentrations of melamine and the various aldehydes (data not shown). Using variable amounts of melamine in the incubation mixtures had no effect on the intensity order of the glycated compounds that were formed with methylglyoxal and DL-glyceraldehyde, once again, producing AGEs with the highest and lowest UV intensities, respectively.  Analysis of the data in Table II showed that with methylglyoxal there were more AGE products formed than with glyoxal and DL-glyceraldehyde, with DL-glyceraldehyde yielding the lowest number of AGEs. A further evaluation of the results in Table II revealed    Other findings revealed that increases in pH promoted melamine AGEs with D-galactose and methylglyoxal ( figure 18). This effect of pH on the glycation of melamine was not surprising since increases in pH have been shown to promote Schiff base formation (15 -16).

Discussion
Melamine is a chemical that has been employed in the manufacture of adhesives, laminates, plastics, floor tiles, kitchenware, and commercial filters. Yet, it has been also used as an additive to artificially boost a food's protein content, creating a lucrative market for edibles believed to be of Melamine alone has been shown to cause no or little toxicity in animals (29 -30); however, when combined with cyanuric acid, the two chemicals can trigger the formation of kidney stones which ultimately leads to renal failure (29 -31).
In 2008, the melamine crisis expanded from a pet problem to a human problem when 'scrap melamine' was discovered in infant's formulas,                           Since the non-enzymatic glycation of proteins is tightly related to the pathology of long-term diseases, the inhibitors to prevent the formation of AGEs have been extensively investigated over years [6][7][8]. The reactants involved in the initial stage of glycation are a common target for anti-glycation drug design [9,10]. This class of inhibitors act as sugar competitors by interacting with target glycation amino acid residues, most commonly lysine and arginine, and prevent their further modification by reducing sugars. Two notable potential anti-glycation agent fall into this class include acetylating aspirin and aldehyde pyridoxal-5'-phosphate.
Previous study in our laboratory showed that 2 nm gold nanoparticles (GNPs) exhibit an inhibitory effect on the formation of AGEs when Human Serum Albumin (HSA) was glycated by DL-glyceraldehyde [11]. GNP typically refers to gold colloid with a particle size ranging from one nanometer to several hundred nanometers. Due to its unique optical property, very large surface-to volume ratio and easy modification, GNP has been applied in a broad range of fields including biomedicine, bio-imaging, material science and synthetic chemistry [12][13][14].
Proteins and amino acids are able to covalently bond to the gold atoms on the surface of nanoparticle through Au-S or Au-NH 2 bonds [15,16]. The binding of biomolecules to GNP can stabilize the particle from conjugation and enhance its solubility in aqueous solution as well [17]. In the meanwhile, free amino groups in proteins or peptides, some of which are the seeding sites of GNP to these biomolecules, might also be reaction sites susceptible to glycation by reducing sugar. This suggests that the specific covalent bonding of GNP to these amino groups in protein might be able to protect these sites from glycation. Furthermore, GNP could possibly bind to the glycation intermediates of protein and prevent the formation of reactive dicarbonyl compounds. By trapping these AGE precursors, GNP could stabilize and fix the protein structure from further alteration and prevent the formation of AGEs.
The objective of this study was to evaluate the rate and extent of BSA glycation by D-ribose in the presence of spherical GNPs. A combination of UV spectrometry, HPLC, circular dichroism and MALDI were used to determine the formation of AGEs. This study emphasized also on the effect of different nanoparticle sizes on GNP's inhibitory efficiency. The correlation between particle size, total particle surface area in solution and GNP's anti-glycation ability was evaluated by two separate experiments designated as experiment A and B. As GNP is highly biocompatible and water soluble, an insight of its anti-glycation effect and its inhibition mechanism on the formation of AGEs might lead to a novel therapeutic application of GNP on reducing AGE related complications.

UV and fluorescence spectroscopy
UV spectrum with wavelength ranging from 200 nm to 400 nm was determined on an UltroSpec 2100 instrument (Biochrom Ltd, Cambridge, UK). Fluorescence measurements were made at respective excitation and emission wavelengths of 340 nm and 420 nm using a Spectra Max M2 spectrometer (Molecular Devices, Sunnyvale, CA, USA). All samples were diluted 50 fold before analysis and all readings were obtained in thermostatically controlled cuvettes that were maintained at 25±1 °C. The above excitation and emission wavelengths were determined optimal for detecting BSA AGEs.

Circular dichroism
CD analysis were performed at a Jasco J-720 spectropolarimeter (Tokyo, Japan) using a quartz cuvette of 1 mm path length. The instrument was controlled by Jasco's Spectra Manager software. Before analysis, the concentration of BSA was adjusted to 0.5 mg/ml using 0.1 M phosphate buffer, pH 7.2. Signals were obtained in the far-ultraviolet region (190-250nm). A total of 10 consecutive scans with bandwidth of 1 nm, scan speed of 20 nm/min and response time of 2 s were averaged. The spectrums were then subtracted by corresponding blanks to eliminate any buffer effect.

High-performance liquid chromatography
Each HPLC run was performed in triplicate using a Hewlett Packard Each spectrum was the sum of 500 single laser shots randomized over 10 positions within the same spot (500/50). Analysis of data was performed using FlexAnalysis and ClinProTools (Bruker Daltonics). c and d to the same mass value of 0.05 mg/ml resulted in a different particle number and total particle surface area in each solution. The value of total particle surface area in unit volume (ml) was obtained by multiplying the particle number in one unit volume (ml) with the single particle surface area (nm 2 ). According to table I, 5 nm GNP solution had the highest total particle surface area in solution, followed by 2 nm GNP and then 20 nm. Interestingly, this sequence corresponded to the order of their anti-glycation efficiency that 5 nm GNP showed the most reduction on the formation of AGEs while 20 nm the least.

Results
To further evaluate the correlation between nanoparticle size, total particle surface area in solution and GNP's anti-glycation ability, we conducted experiment B. In this separate study, we used the same blank and control samples as experiment A, while adjusted the Au mass concentration to 0.02 mg/ml, 0.01 mg/ml and 0.05 mg/ml for 2 nm GNP, 5 nm GNP and 20 nm GNP solutions respectively, resulting in a same particle surface area in all three GNP solutions. Figure 2  Advanced glycation of BSA not only produces aromatic AGEs that can be detected by UV absorbance, but also alters proteins secondary structure whose change can be monitored by circular dichroism in the far-UV spectral region (190-250 nm). The secondary structure of BSA is composed of about 67% α-helix structure, so BSA conformation change was determine by monitoring the loss of α-helix structure in each sample. Figure 3 shows CD spectrum profiles for experiment A in the far-UV spectral region and figure 4 shows the CD data for samples in experiment B. The CD spectrum for intact GNPs also revealed decreases in the loss of secondary structure comparing to control BSA which was glycated without GNP. CD spectrums for samples in experiment A revealed that reaction mixture with 5 nm retained the most helical structure followed by 2 nm sample then 20 nm. In contrast, CD profiles for experiment B seen in figure 4 showed that all three sizes of nanoparticle exhibited similar anti-glycation ability, producing CD spectrum almost superimposable to each other. GNP solution with enhanced total surface area exhibited a higher inhibitory effect against the formation of AGEs. This anti-glycation property of AGEs was reduced by decreasing the nanoparticle numbers in solution which resulted in a decrease of total particle surface area.

Discussion
The The other important finding of this study was the correlation between GNP's anti-glycation efficiency and the total nanoparticle surface area in the solution. GNP's inhibitory effect against the formation of AGEs could be enhanced by increasing the total GNP particle surface areas in solution. This can be achieved by increasing the total nanoparticle concentration or increasing the nanoparticle sizes. It should be noted that larger size GNP or saturated GNP solution tends to conjugate with protein and precipitates out.
Thus it is important to keep the particle size and concentration within a range which GNPs still retain their nanoparticle properties without aggregating with protein during glycation. In our study, we found that 5 nm GNPs with an Au mass concentration of 0.05 mg/ml exhibited the most anti-glycation ability while stayed homogenously during the entire incubation process.
The mechanism of GNP's inhibitory effect against the formation of AGEs is still under investigation. We proposed that GNPs competitively bind to the potent glycation sites on BSA, preventing their further modification by glycation. There is a strong implication that amino acid residues are able to covalently bond to the gold atoms on the surface of nanoparticle through Au-S or Au-NH 2 bonds. Such binding not only stabilizes nanoparticle from conjugation, but also protects free amino groups such as lysine from glycation. Furthermore, GNP could possibly bind to the glycation intermediates of protein and prevent the formation of reactive dicarbonyl compounds.
In the specific case of our study, GNP with a higher surface area exhibited better inhibitory ability against glycation, probably due to the increase in available binding area on GNP surface to protein's potential glycation sites. As GNP is highly biocompatible and water soluble, an insight of its anti-glycation effect and its inhibition mechanism on the formation of AGEs might lead to a novel therapeutic application of GNP on reducing AGE related complications. The total nanoparticle surface area in each solution was adjusted to the same value (refer to table I). The spectrum is ranging from 250 nm to 400 nm with glycation products absorbed most at 280 nm. All data points represent the averages of three replicate measurements.

Introduction
Human serum albumin (HSA) constitutes almost half of the protein content in the plasma of normal healthy individuals [1]. As one of the major circulating protein in blood, HSA is the most abundant protein and utilizes a wide variety of physiological functions due to the flexibility of its structure [2].
First of all, albumin has a comparatively low molecular weight of 66 KDa comparing to other globulins in the plasma system. This property makes the protein a key element in maintaining the osmotic pressure needed for proper distribution of body fluids between intravascular compartments and body tissues [3]. HSA also functions as a carrier protein for small size metabolites such as hydrophobic steroid hormones, ions and fatty acids.
The binding sites which allow the affinity for these metabolites are distributed widely on all three domains of the protein. As a result, albumin is also involved in the pharmacokinetics of many therapeutic drugs that can be bound to the protein [4,5]. In addition, HSA represents the predominant antioxidant in plasma, a body compartment continuously exposed to high oxidative stress [6,7]. Previous study has shown that more than 70% of the free radical-trapping activity of serum was due to HSA [6].
In normal conditions, HSA has a long half-life time of about 21 days and a high plasmatic concentration between 35 to 50 mg/ml. Being constantly exposed to numerous metabolites, albumin is highly sensitive to the structural modification by these molecules. In particularly, non-enzymatic glycation is among the major mechanisms which contribute to the alteration of HSA's functions. Glycation is a non-enzymatic process that involves a series of steps initiated with a condensation reaction between the aldehyde group of a reducing sugar and the free amino group of a protein, resulting in the formation of an unstable aldimine, also known as the Schiff base. This intermediate then undergoes Amadori rearrangement and converts to a more stable ketoamine structure referred to as Amadori products. Late stage of glycation involves irreversible modification of these early stage glycation intermediates such as dehydration, cyclization, oxidation, and rearrangement to form a polymorphic group of compounds called advanced glycation endproducts (AGEs) [8][9][10]. The accumulation of AGEs plays a significant role in the pathogenesis of diabetic complications.
Glycated albumin accounts for about 80% of the circulating glycated protein [11]. In vivo, the proportion of glycated albumin in healthy persons is in the range of between 1% and 10% [2]. In the case of diabetes mellitus, the proportion of glycation HSA may increase two to three fold and even reach to 90% for severe diabetic patients with poor diabetic control [12,13].
Glycation-induced modifications have a determinant impact on HSA's biological functional properties such as its antioxidant property and metabolites binding capacity. In addition, glycation is accompanied by the production of AGEs, which triggers the release of pro-inflammatory molecules and free radicals, inducing oxidative cellular dysfunction.
The glycation level of HSA can be up-graded by several factors including genetic deficiency, high calories diet and environmental factors.
UV light is electromagnetic radiation with a wavelength in the range of 100 nm to 400 nm. UV radiation is classified into three types or bands-UVA (400-315 nm), UVB (315-280 nm), and UVC (280-100 nm). Exposure to ultraviolet (UV) irradiation is known to perturb protein structure and generate reactive oxygen species (ROS) [14,15]. Solar UVC is absorbed by ozone in the stratosphere and the biological effect on human beings from natural sources is limited. However, with increasing use of man-made UVC radiation such as germicidal application and light therapy for wound healing, there is a growing concern about the biological effect of UVC. Previously study has shown that UVC irradiation could disturb structural stability of HSA and cause conformational rearrangement and aggregation of the protein [16].
The production of ROS was also found to be stimulated by UVC irradiation in vivo [17].
ROS play a significant role in the mechanism of protein glycation. The

Preparation and analysis of intact HSA by MALDI-TOF MS
All samples were purified with C 4 ZipTips and analyzed on a Bruker Autoflex MALDI-TOF (time-of-flight) mass spectrometer (Bruker Daltonics, Billerica, MA, USA). Prior to analysis, the protein samples (0.6 μl) were mixed with a 50% aqueous acetonitrile solution (0.6 μl) of saturated sinapinic acid containing 0.05% trifluoroacetic acid (TFA) as matrix, spotted onto a stainless steel sample plate, and allowed to air dry. Spectra were acquired in linear TOF mode with a 550 ns delay in the m/z range from 20,000 to 80,000.
Each spectrum was the sum of 500 single laser shots randomized over 10 positions within the same spot (500/50). Analysis of data was performed using FlexAnalysis and ClinProTools (Bruker Daltonics).

Detection of aromatic AGEs by UV spectrometry
Tables I shows the UV absorption readings of incubation mixtures containing 35 mg/ml HSA with 20 mM D-glucose (hereinafter referred to as "HSA-glc") in both experiment A and B. In experiment A, samples were collected at different time points (0, 1, 2, 4 and 7 days of incubation respectively). In both control sample which was incubated in dark and experimental sample which was exposed to constant UVC radiation, HSA was found to form AGEs and the highest levels of glycation were achieved when HSA reacted with D-glucose for 7 days. It should be noted that a severe protein crosslinking was observed in UV exposed sample after 7 nm revealed two important findings: 1) UVC radiation enhanced the formation of aromatic AGEs of HSA, which was found to be responsible for the increased UV intensity at 280 nm; 2) HSA exposed to UVC radiation was more prone to be glycated by reducing sugar. This finding suggested that UVC treatment might cause conformational restructuring of HSA, making the protein more susceptible to glycation.

Quantification of AGEs by HPLC
The extent of HSA glycation under the treatment of UVC radiation in both experiment A and B was determined by HPLC. Figure 1   According to the peak area intensities of these glycation products numbered peak 1 (R t =1.87 min), peak 2 (R t =3.15 min) and peak 3 (5.10 min), experimental sample under UVC radiation yielded AGE peaks with higher band areas comparing to that of control solution incubated in dark (data shown in Table II)  (seen in figure 9). Whereas profile of untreated HSA incubated with D-glucose for 48 hours indicated the formation of only three AGE species formed, a result indicated that more AGE species were formed when HSA was pre-treated with UVC radiation for 24 hours. Moreover, the peak intensity of AGE species in UVC treated solution was higher than the value of untreated control sample, verifying that exposure to UVC radiation made HSA more susceptible to glycation by reducing sugar.

The detection of CML by ELISA
CML is an AGE resulted from oxidative stress and chemical glycation.
CML adducts was found accumulated with aging and over produced in several diabetic related complications. CML-modified proteins are recognized by the receptor RAGE which is expressed on a variety of cells.
This process could initiate the expression of ROS and induce oxidative stress. The formations of CML in HSA-glc mixtures in both experiment A and B were evaluated by ELISA assy. Higher levels of CML leaded to increased CML-antibody conjugate binding and greater OD signal at 450 nm. Figure 10 shows  figure 11). The difference in CML levels between control solution and UVC exposed sample started to be observed at the very early stage of glycation after only 12 hours of incubation.
After 48 hours, UVC treated HSA produced about double amount of CML comparing to control solution, an observation confirming our earlier conclusion base on UV spectrometry and HPLC elution profile.

Measurement of HSA mass shift by MALDI-TOF
To determine the number of sugar addicts on HSA forming by glycation, incubation. In solution incubated in dark, one D-glucose addict was found on HSA after 2 days reaction and two sugar molecules were found to addict on HSA in UVC treated sample. The difference was enhanced by prolonging the incubation time to 7 days. In samples incubated for 7 days, we noticed a mass shift of 740 in solution exposed to UVC radiation, which equals to 8 D-glucose addicts, whereas only 3 sugar molecules were found to addict on HSA in mixture incubated in dark. This observation again corresponded to our early conclusion that UVC irradiation enhanced the degree of glycation.
Moreover, HSA pre-incubated under UVC radiation for 24 hours in experiment B yielded mass shift of 290 after 48 hours incubation, which equals to 3 D-glucose addicts on HSA. Interestingly, this number is closed to the mass shift produced by HSA-glc mixture incubated in dark for 7 days, which suggested that the exposure to UVC not only enhanced the glycation level, but also increased the reaction rate.

Conclusion
The non-enzymatic glycation of HSA has been found to play a major role in human physiology. The reactive carbonyl groups of reducing sugars condensed with the amino groups of certain amino acid residues such as lysine and arginine on HSA and form Amadori products that can rearrange to form AGEs after a variety of chemical reactions. Accumulation of AGEs has been reported to account for many of the chronic complications including diabetes and Alzheimer's disease.
Recently, there has been an increasing interest on the physiological effect of UVC radiation since they are readily absorbed by DNA and are highly genotoxic [19]. The biological effects of ultraviolet radiation (UV) include DNA damage, mutagenesis, cellular aging, and carcinogenesis.
These effects are in part mediated by reactive oxygen species (ROS), the production of which is stimulated by UV irradiation of cells and tissues [16,18]. Since the production of ROS also enhanced the glycation level in biological system, current study made an effort to evaluate the influence of UVC radiation on the formation of AGEs when physiological concentration of HSA was glycated by D-glucose in-vitro.
A combination of several analytical methods including UV spectrometry, HPLC and MALDI-TOF revealed that UVC radiation enhanced the glycation level of HSA even at the early stage of the reaction. Moreover, the glycation rate was also boosted when the reaction took place under UVC exposure.
HSA started to form noticeable cross-link structures after a few days treatment with UVC, denaturized, and precipitated out from the reaction system. HSA pre-incubated under UVC radiation was more susceptible to glycation by reducing sugar comparing to untreated HSA. Although the detailed mechanism of this phenomenon is still under elucidation, it might be related to the alteration in HSA's conformational structure when the protein is exposed to UVC radiation.
In this study we also quantified the production of CML from HSA glycation using ELISA immune assay. The major source of CML during protein glycation is oxidative degradation of Amadori intermediate and ROS was found to accelerate the formation of CML. We observed a considerable increase in CML production when the protein was glycated under UVC radiation; a finding suggested that UVC radiation promoted the formation of ROS, which could then stimulate oxidative degradation of Amadori addicts to form AGEs.
At present, there is little information on the biological effect of short UV wavelength, and there is practically no data on glycation of HSA under the influence of UVC radiation. UVC radiation was found to enhance the glycation rate and promote the formation of HSA AGEs. The high energy UV radiation was also suspected to perturb protein structure; making protein more susceptible to glycation. As a result, more attention on the safe usage of UVC light should be addressed because the negative biological effect of high energy, short wavelength UV may not only be related to their genotoxic but may also be caused by their ability to enhanced protein glycation.