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Glycobiology vol. 24 no. 11 pp. 979–990, 2014 doi:10.1093/glycob/cwu057 Advance Access publication on June 19, 2014 REVIEW Glycoxidation of biological macromolecules: A critical approach to halt the menace of glycation Saheem Ahmad1,2, M Salman Khan2, Firoz Akhter2, Mohd Sajid Khan1,2, Amir Khan3, J M Ashraf4, Ramendra Pati Pandey5, and Uzma Shahab1,6,7 2 Department of Biosciences, Integral University, Lucknow, India; 3Glocal School of Life Sciences, Glocal University, Saharanpur, Uttar Pradesh, India; 4 Department of Biotechnology, School of Biotechnology, Yeungnam University, Yeungnam, Republic of Korea; 5Nano-Biotech Lab, Department of Zoology, Kirorimal College, University of Delhi, Delhi, India; and 6Department of Biochemistry, Central Drug Research Institute, Lucknow, India Received on November 3, 2013; revised on May 29, 2014; accepted on June 12, 2014 Glycation is the result of covalent bonding of a free amino group of biological macromolecules with a reducing sugar, which results in the formation of a Schiff base that undergoes rearrangement, dehydration and cyclization to form a more stable Amadori product. The ﬁnal products of nonenzymatic glycation of biomacromolecules like DNA, proteins and lipids are known as advanced glycation end products (AGEs). AGEs may be generated rapidly or over long times stimulated by distinct triggering mechanisms, thereby accounting for their roles in multiple settings and disease states. Both Schiff base and Amadori glycation products generate free radicals resulting in decline of antioxidant defense mechanisms and can damage cellular organelles and enzymes. This critical review primarily focuses on the mechanistic insight of glycation and the most probable route for the formation of glycation products and their therapeutic interventions. Furthermore, the prevention of glycation reaction using therapeutic drugs such as metformin, pyridoxamine and aminoguanidine (AG) are discussed with special emphasis on the novel concept of the bioconjugation of these drugs like, AG with gold nanoparticles (GNPs). At or above 10 mM concentration, AG is found to be toxic and therefore has serious health concerns, and the study warrants doing this novel bioconjugation of AG with GNPs. This approach might increase the efﬁcacy of the AG at a reduced concentration with low or no toxicity. Using the concept of synthesis of GNPs with abovementioned drugs, it is assumed that toxicity of various drugs which are used at high doses can be minimized more effectively. 1 To whom correspondence should be addressed: Tel: +91-9452677867; Fax: +91522-2890809; e-mail: [email protected] (S.A.); Tel: +91-522-2890730; Fax: +91-5222890809; e-mail: [email protected] (M.S.K.); Tel: +919450612013; Fax: +91-5222257539; e-mail: [email protected] (U.S.) 7 Present address: Department of Biochemistry, King George Medical University, Lucknow, U.P., India. Keywords: advanced glycation end products / antiglycation / gold nanoparticle / oxidative stress / therapeutic intervention Introduction The story of glycation reaction was started way back in the year 1912 when Louise Camille Maillard ﬁrst described the glycation reaction after whom the reaction is also known as the Maillard reaction (Maillard 1912). Glycation is the process whereby sugars bind to the free amino residues of proteins, lipids and DNA macromolecule. Sugars and other reactive carbonyl compounds bind spontaneously to nucleophilic amino groups of amino acids and proteins in a nonenzymatic process. Reducing sugars, such as glucose in basic solutions and lipids by β-oxidation or peroxidation generate formyl (an aldehyde) and ketone groups. Aldehydes and ketones have a highly polarized carbonyl (C=O) group, the oxygen atom of which is electronegative and may react with nucleophiles in proteins and other biomolecules like, DNA and lipids. Under high glucose load (hyperglycemic condition), these biomolecules undergo a nonenzymatic glycation reaction leading to the formation of a complex series of compounds known as the advanced glycation end products (AGEs). This, in turn, results in the deprivation of the functions of the biological macromolecules by altering their structural conformation. Figure 1 schematically represents the probable pathway of reaction of biomacromolecules with reducing sugars to form protein AGEs, advanced lipoxidation end products (ALEs) and DNA advanced glycation end products (DNA-AGEs). Glycation and oxidative stress are closely linked and are often referred to as “glycoxidation” process which is believed to be involved in the complications associated with several disorders including diabetes, cardiovascular disease and Alzheimer’s disease, in addition to various forms of cancer (Aldini et al. 2013). Glycation of biomacromolecule DNA glycation When DNA reacts with sugars in vitro at a physiological temperature, the formation of DNA-bound AGEs is observed (Mustafa et al. 2012; Ahmad et al. 2014). Glycation of DNA has shown to considerably alter the structure of DNA macromolecule and it leads to depurination, strand breaks and mutations such as insertions, deletions and transposition (Ahmad, Ahmad, et al. 2011; Ahmad, Moinuddin, et al. 2011). Therefore, DNA-AGEs could contribute to the loss of genomic integrity, which occurs during © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected] 979 S Ahmad et al. Fig. 1. Schematic representation of probable pathway of macromolecules reacting with reducing sugars to form AGEs/ALEs and DNA-AGEs, respectively. This Figure was adapted from our published research paper in the Elsevier journal “International Journal of Biological Macromolecules” (2013), 58:206–210. The permission is automatically granted to the authors/corresponding authors of the paper as per Elsevier STM Permission Guidelines (2012). aging and may contribute to the age-related complications. More detailed studies on the stability and dynamics of DNA showed that glycation leads to partial unwinding and/or fragmentation of the double helix (Mustafa et al. 2012). Wuenschell et al. (2010) investigated the mutagenic potential of the predominant DNA-glycation adduct carboxy ethyl deoxygunaosine (CEdG) and exhibited that CEdG within the template DNA and the corresponding triphosphate possess different syn/anti conformations during replication which inﬂuence base pairing preferences. Oxidative modiﬁcations within the DNA lead to reduced gene expression (Nagai et al. 2010). We have shown in the recent past that genotoxicity and immunogenicity are incurred in DNA and proteins by carcinogens and reactive oxygen species (ROS) as well (Moinuddin et al. 2012; Shahab, Ahmad, et al. 2012; Shahab, Moinuddin, et al. 2012; Shahab et al. 2013). Moreover, most recently, our research group has also shown that glycation-induced oxidative stress leads to the modiﬁcation of DNA macromolecule and results in alteration of its structure (Akhter et al. 2013). The structural 980 perturbations in the DNA macromolecules are the consequence of the genotoxic effect of the glycation reaction (Ahmad, Moinuddin, et al. 2011). The structural pathway for the formation of DNA bases Amadori products is shown in Figure 2. In vitro incubation of DNA with glucose, fructose and glucose-6-phosphate led to UV absorbance and ﬂuorescence changes, indicating that DNA undergoes nonenzymatic browning reaction (Ahmad, Ahmad, et al. 2011; Ahmad, Moinuddin, et al. 2011). So far, it has been shown that among all DNA bases, deoxyguanosine exhibited the highest glycation rate with reactive carbonyl species (RCS) (Li et al. 2008). Therefore, free guanosine, guanine or 2′-deoxyguanosine were used in model incubations with sugar or RCS to identify the structure of possible DNA adducts. N2-carboxyethyl-2′-deoxyguanosine (CEdG A,B) was established as a sensitive marker for DNA modiﬁcations by the carcinogenic substance methylglyoxal (MG). The role of DNA-glycation in vivo has been discussed in great detail in our recent publication where we showed the Glycation and its inhibition by nanoconjugation Fig. 2. Mechanistic representation of glycation reaction between reducing sugars (ribose, glucose and deoxyribose) and adenine/guanine base. presence of autoantibodies in type 1 and type 2 diabetes patients against glycated human DNA (Ahmad et al. 2014). Elevated levels of AGEs have been implicated in the pathological complications of diabetes, uremia, Alzheimer’s disease and possibly cancer. CEdG adducts were speciﬁcally detected in a human breast tumor and normal adjacent tissue at levels of 3–12 adducts/107 dG, suggesting that this lesion may be widely distributed in vivo which is an adduct of DNA glycation only. Protein glycation The protein glycation is an inevitable, nonenzymatic reaction between reducing sugars and proteins, occurring in all living systems. Although the reaction is slow, it is quite dynamic in nature and starts with the formation of unstable Schiff base, which undergoes a series of reactions leading to the formation of heterogeneous molecules called AGEs (Thornalley et al. 1999). Glucose and glucose-derived dicarbonyls such as glyoxal, MG, glucosone and 3-deoxyglucosone (3-DG) are the major precursors of AGEs. The levels of these precursors determine the formation of different types of AGEs. The predominant AGE modiﬁcations include fructosyl-lysine, carboxymethyl lysine (CML), carboxyethyl lysine (CEL) and pentosidine. AGEs are accumulated in the body due to in vivo glycation as well as through intake of exogenous AGEs, which are mainly formed due to overheating of foods and beverages (Faist and Erbersdobler 2001). Diabetes promotes the formation of AGEs in vivo, thus, enhancing the overall accumulation. AGEs cause cell damage at various levels, namely (i) alteration of protein structure and function; (ii) protein aggregation, ﬁbril formation and protease resistance (Wei et al. 2012); (iii) aberrant signaling through interaction with the RAGE and (iv) dysfunction of extracellular matrix. AGEs contribute substantially to the progression of diabetic complications, including nephropathy, retinopathy, neuropathy; cardiovascular diseases, cataract, accelerated aging, neurodegenerative diseases and cancer. Studies of protein glycation have focused on the reaction of aldoses and ketoses, particularly glucose, with lysine residues of proteins. The acyclic form of the monosaccharide reacts reversibly with the lysyl side chain of amino group to form an initial Schiff base adducts. This exists mainly in the cyclic glycosylamine form (Neglia et al. 1983). AGEs are formed slowly 981 S Ahmad et al. throughout life and the concentrations of AGEs found represent a lifelong accumulation of the glycation adducts. This applies to chemically stable AGE residues formed on long-lived proteins. For example, CML, CEL and pentosidine residue accumulation on skin collagen (Shimasaki et al. 2011). Various proteins and enzymes of clinical signiﬁcance as well as those involved in cellular processes such as glucose metabolism, bioenergetics, cell repair and stress response have been affected by glycation. Plasma proteins especially human serum albumin (HSA), immunoglobulins, apolipoprotein, ﬁbrinogen and transferrin are predominantly glycated and implicated in various disease conditions (Zhang et al. 2008). Hemoglobin undergoes extensive glycation and is an established biomarker for diabetes (Lyons and Basu 2012). Likewise, early glycation products of albumin could serve as markers for secondary complications of diabetes. Glycation of albumin decreases the antioxidant activity and binding capacity (Rondeau and Bourdon 2011). Similarly, AGE modiﬁed immunoglobulin G presents unique neoepitopes, which elicit autoimmune response in rheumatoid arthritis patients (Ahmad et al. 2012). Increased glycation of apolipoproteins may play a role in the accelerated development of atherosclerosis. Glycation of ﬁbrinogen alters the formation of ﬁbrin network kinetics, which contributes to decreased pore size and lysis rate of ﬁbrin clots (Pieters et al. 2008). Transferrin glycation is associated with increased free radical production, lipid peroxidation and decreased iron-binding capacity (Van Campenhout et al. 2003). It has also been found that glycation affects calcium signaling and calcium-dependent processes. Human paraoxonase 1, a calcium-dependent esterase responsible for metabolism of membrane lipid hydroperoxides, is inactivated by glycation and implicated in coronary heart disease (Mastorikou et al. 2008). Other proteins such as human complement regulatory protein show reduced activity due to glycation, thereby promoting membrane attack complex formation in the target organs of diabetic complications (Qin et al. 2004). Glycation modiﬁcation results in the diminished enzyme activity of creatine kinase, alanine aminotransferase and cytoplasmic aspartate aminotransferase aiding in the development of diabetic complications (Beranek et al. 2001). Several intracellular enzymes especially those involved in glucose metabolism have been greatly affected by glycation modiﬁcations. Key enzymes such as glyceraldehyde 3-phosphate dehydrogenase, bisphosphoglycerate mutase from erythrocytes and pancreatic glucokinase are inactivated by glycation (Zhao et al. 2000). Increased glycation of glycolytic enzymes leads to higher accumulation of carbonyls, which may further modify proteins. Other enzymes which show loss of activity upon in vitro glycation include enolase, nitric oxide synthase, catalase and Cu/Zn superoxide dismutase (SOD1) (Pietkiewicz et al. 2009). In vitro glycation of Na-KATPase at different amino groups has shown differential catalysis and cation binding, suggesting that glycation not only inhibits enzyme activity but also modulates enzyme kinetics (Garner et al. 1990). Interestingly, glycation-induced loss of functional activity is compensated by increased gene expression. One of the well-studied examples is the decreased functional activity of glutathione system due to carbonyl stress, which is compensated by increased expression of gamma-glutamylcysteine synthetase enzyme (Miyahara et al. 2002). Nevertheless, targeting any of the enzyme or protein involved in the nonenzymatic glycation 982 may prevent their ultimate structure consequently their function as well. The need of the hour is to stop this slow and steady glycation reaction with minimal or no toxicity. The approaches used in the past have shown some deleterious effects on humans undergoing trial in diabetic nephropathy patients against aminoguanidine (AG). Lipoproteins glycation In diabetes, hyperglycemia induces modiﬁcation of plasma and tissue proteins by nonenzymatic glycation, a gradual process that culminates with the formation of irreversible AGE. AGE accumulates particularly at sites of atherosclerotic lesions, but the mechanisms whereby AGE contributes to diabetes-induced accelerated atherogenesis are not fully understood. Low-density lipoproteins (LDLs), either oxidized or glycated, are present in the plasma and in the affected vasculature of diabetic patients (Virella et al. 2003). Although there are several studies on the effect of glycated proteins on vascular cells (Younis et al. 2008), only few data exist on the effect of irreversibly glycated LDL (AGE-LDL) on endothelial cells. AGE interact with speciﬁc cellular receptors, the best characterized of which is the receptor for AGE (RAGE). Interestingly, elevated levels of AGE-LDL are present in the sera of euglycemic or normolipemic patients with atherosclerosis. AGE-LDL is present in the vessel wall atheroma and exerts proatherogenic effects (Sima and Stancu 2002; Hodgkinson et al. 2008). It has been found that recognition of glycated LDL by LDL receptor is impaired, while the uptake by monocytemacrophages are enhanced, which contributes to hyperlipidemia and accelerated foam-cell formation, respectively (Toma et al. 2009). It has been hypothesized that in diabetes, hyperglycemia and the ensuing increased formation of AGE-LDL may directly affect the vascular endothelium by activating RAGE, which in turn may induce a series of changes leading to a prooxidant and pro-inﬂammatory state, ﬁnally generating endothelial dysfunction characteristic for micro- and macroangiopathy. It has also been reported that AGE-LDL induce in human endothelial cell (HEC) an increased expression of RAGE, p22phox, p67phox and NOX4, leading to an increase in NADPHox activity, and an augmented expression of monocyte chemoattractant protein-1. High glucose augments the effect of both nLDL and AGE-LDL on HEC (Toma et al. 2009). Moreover, the glycation of lipoproteins in diabetes was ﬁrst reported by Schleicher et al. (1981). It has been reported that in healthy nondiabetic people the serum concentration of glycated apolipoprotein B (apo B), although lower than in diabetic people was on average 5 mg/dl, representing 4% of the total apo B (the major component of the protein moiety of LDL) (Tames et al. 1992). It has been reviewed the possibility that glycated lipoproteins, including very-low-density lipoprotein, LDL and also HDL could contribute to atherosclerosis. It has been consistently observed both in tissue culture and during in vivo turnover studies that glycated LDL is not cleared by the physiological LDL receptor (Wang et al. 1998). It thus has a slower catabolic rate than nonglycated LDL. Glycation of LDL apo B involves epitopes close to its receptor-binding site, suggesting that a conformational change in the binding site inﬂuences recognition Glycation and its inhibition by nanoconjugation by LDL receptors. Glycated LDL is thus more likely to be cleared by scavenger receptors on macrophages and endothelial cells, to which its binding is not compromised by glycation, and subclasses of scavenger receptors speciﬁc for AGEs have been described (Schmidt et al. 1999). Furthermore, glycation and oxidation are by no means mutually exclusive naturally occurring modiﬁcations of LDL, because glycation itself generates free radicals (glycoxidation) (Jenkins et al. 2004). Glycoxidated LDL is present in the atheromatous plaque (Imanaga et al. 2000). It has, however, been shown in vitro that prior oxidation is not essential for glycation of LDL to occur (Li et al. 1996). Advanced glycation end products: source and target AGEs are formed endogenously when the carbonyl groups of reducing sugars nonenzymatically react with the free amino groups on proteins, lipids and DNA. AGEs are generated in vivo as a normal consequence of metabolism, but their formation is accelerated under conditions of hyperglycemia, hyperlipidemia and increased oxidative stress. Although glucose is relatively slow in reacting with proteins, highly reactive dicarbonyl compounds (generated as a result of glucose autooxidation, lipid peroxidation and the interruption of glycolysis by reactive oxygen species) are capable of rapid AGE formation. Dicarbonyls such as glyoxal, MG and 3-DG interact with intracellular proteins to form AGEs, and can also diffuse out of the cell and react with extracellular proteins. Excessive AGE accumulation results in signiﬁcant cellular dysfunction by inhibiting communication between cells, altering protein structure and interfering with lipid accumulation within the arterial wall (Barlovic et al. 2010). AGEs are well reported to bind to the RAGEs which activates nuclear factor kB, triggering oxidative stress, thrombogenesis, vascular inﬂammation and pathological angiogenesis (Yamagishi et al. 2007), thereby contributing to many of the long-term complications of diabetes. More recently, AGEs have been implicated in the pathogenesis of type 2 diabetes by contributing to the development of insulin resistance and low-grade inﬂammation known to precede the condition (Tahara et al. 2012). Apart from endogenous AGE formation, AGEs and their precursors are also absorbed by the body from exogenous sources such as cigarette smoke and through consumption of highly heated processed foods. Browning of food during cooking is used to enhance the quality, ﬂavor, color and aroma of the diet. This process (known as the Maillard reaction) generates large quantities of AGEs. Factors that enhance AGE formation in foods include high lipid and protein content, low water content during cooking, elevated pH and the application of high temperature over a short time period. More AGEs are generated in foods exposed to dry heat (grilling, frying, roasting, baking and barbecuing) than foods cooked at lower temperatures for longer time periods in the presence of higher water content (boiling, steaming, poaching, stewing or slow cooking) (Uribarri et al. 2005). Kinetic studies have demonstrated that 10–30% of dietary AGEs consumed are intestinally absorbed (Faist and Erbersdobler 2001), with only one-third of ingested AGEs excreted in urine and feces. Plasma AGE concentration appears to be directly inﬂuenced by dietary AGE intake and the body’s capacity for AGE elimination (Delgado-Andrade et al. 2012). Low-AGE diets in animal studies have been shown to reverse insulin resistance and chronic inﬂammation, inhibit the progression of atherosclerosis and prevent experimental diabetic nephropathy and neuropathy (Uribarri et al. 2007), but whether these results can be translated to humans is uncertain. Cross-sectional and case–control studies involving humans with impaired renal function or diabetes have demonstrated associations between elevated AGE intakes and serum biomarkers of oxidative stress, endothelial dysfunction, inﬂammation, hyperlipidemia and hyperglycemia (Chao et al. 2010). AGEs have also recently been implicated in the dysfunction and death of pancreatic beta cells, leading to the hypothesis that excessive AGE formation and oxidative stress possibly have a role in the development of type 1 and type 2 diabetes (Coughlan et al. 2011). Low-AGE diets have been suggested as a possible future therapeutic option for healthy individuals at risk for the development of type 1 or type 2 diabetes (Vlassara and Striker 2011). Through reduced consumption of highly processed heattreated foods, dietary AGE restriction may represent a relatively simple, noninvasive therapy for the effective treatment of many of the metabolic disturbances attributed to excessive AGE levels. This competent review might help to better understand to stop the glycation menace either by dietary AGEs restriction as reviewed here or by using novel therapeutic drugs and novel bioconjugation approach discussed elsewhere. Interrelations of ROS, RCS and AGEs The nonenzymatic glycation of DNA or protein is the process which links chronic hyperglycemia to a series of pathophysiological alterations and considered important in the development of diabetes and the associated diseases (Ahmad, Shahab, et al. 2013). AGEs are direct pathogenic and accumulate in the plasma, serum and tissues of patients in different diseases, e.g. diabetes, end stage renal disease, cardiovascular, aging and arthritis (Peppa et al. 2003). The excess free radical production under hyperglycemic conditions originates from mitochondrial respiration, cytochrome p450, xanthine oxidase, PKC dependent activation of NADH/ NADPH oxidase and RAGE-triggered cellular oxidant stress (Bandeira et al. 2013). Under hyperglycemic conditions several pathways gets activated, namely (i) autooxidation of glucose which leads to the actual glycation reaction thus forming ROS species, (ii) the generation of sorbitol pathway, (iii) activation of PKC pathway by diacyl glycerol generation (DAG), (iv) activation of glucosamine and (v) mitochondrial pathway. All the above pathways ultimately lead to the formation of ROS and result in deleterious effects on protein, DNA and LDL macromolecule. Figure 3 shows the free radical formation and the check points where this slow and steady can be stopped. Furthermore, ROS, within certain boundaries, is essential to maintain homeostasis. Most recently, new sources of ROS generation have been reported (Brieger et al. 2012). These ROS species are also generated in the early and the advanced glycation processes and these species have been shown to exhibit cytotoxicity (Figure 4). During the rearrangement process of the glycation reaction there is also the generation of free radicals like ·OH and O·2− (Ahmad, Moinuddin, et al. 2011; Ahmad, Akhtar, et al. 2013). 983 S Ahmad et al. Fig. 3. Schematic representation of the formation of ROS and RNS under hyperglycemic condition: probable check points “×” to control the reaction. Carbonyl stress is an imbalance of RCS production and carbonyl scavenging mechanisms, which originate from a multitude of mechanistically related pathways, such as glycation, autooxidation of sugars, amino acid metabolism, lipid peroxidation and UV damage (Sergei et al. 2008). The formation of RCS during glycation reaction is shown in Figure 4. Generation of reactive intermediate products is an important step in the glycation. These compounds are known as α-dicarbonyls and include 3-DG, glyoxa and MG. 3-DG rapidly reacts with protein amino groups to form AGE such as imidazolone, pyrraline and CML (Jono et al. 2004). MG may be produced by nonenzymatic pathways from spontaneous decomposition of triose phosphates, autoxidation of carbohydrates and glucose degradation (Nagaraj et al. 2002). In addition to reaction with arginine residues to form imidazolone adducts, MG reacts with lysine residues in protein to form CEL and the imidazolium cross-link, MG-lysine dimer. On the other hand, production of GO in vivo under physiological conditions can yield number of AGEs such as CML, pentosidine, glyoxal-lysine dimer and other nonﬂuorescent AGEs (Nagai et al. 2012). α-Oxoaldehydes are metabolized and inactivated by enzymatic conversion to the corresponding aldonic acids, catalyzed by the glutathione-dependent glyoxalase system (Birkenmeier et al. 2010). 984 Therapeutic intervention Synthetic AGE inhibitors The prevention of glycation reaction is one of the strategies to reduce AGEs. Amadori product formation can be inhibited by compounds that react with the reducing sugars, thereby inhibiting the reaction. The trapping of the reactive carbonyl intermediates formed in the ﬁrst stage of Maillard reaction can be carried out by using guanidine compounds such as AG. Aminoguanidine was the ﬁrst AGE inhibitor proposed to scavenge the dicarbonyl compounds such as glyoxal, MG and 3-DG (Brownlee et al. 1986). Moreover, AG is an investigational drug for the treatment of diabetic nephropathy that is no longer under development as a drug. Numerous in vitro experiments and animal studies conducted on experimental diabetes with AG have shown the prevention of diabetes-induced deteriorations of cardiovascular structure and function and of major long-term complications such as nephropathy, retinopathy and neuropathy (Brownlee et al. 1986; Hammes et al. 1991; Edelstein and Brownlee 1992). Owing to safety concerns resulting from its additional effect, e.g. prooxidant activities and inhibition of NO synthase (Suji and Sivakami 2006), it is currently unlikely that AG will be used to treat diabetic complications (Turgut and Bolton 2010). Most of the studies were comprised of the concentrations ranging from 10 to 100 mM and Glycation and its inhibition by nanoconjugation Fig. 4. Schematic representation of the formation of early and advanced glycation end products. Probable check points “×” to control the reaction was found to be toxic therefore has serious health concerns. Moreover, due to the toxic and prooxidant behavior of the AG, further trials on AG were stopped because of this major apprehension. Moreover, the clinical phases in the AG treatment of patients with type-1 diabetes were also ended due to serious complications in those patients. Another class of AGE inhibitors, “Amadorin” (the postAmadori inhibitors), inhibits the conversion of Amadori intermediates to AGE (Khalifah et al. 1999). The ﬁrst Amadorin identiﬁed was pyridoxamine (PM) that showed a great potential for treatment of diabetic nephropathy. It inhibits AGE formation at different levels by scavenging carbonyl products of glucose and lipid degradation, sequestering catalytic metal ions, blocking oxidative degradation of Amadori intermediate and trapping of ROS. Lalezari-Rahbar (LR) compounds are potent Cu2+ chelators, which inhibit post-Amadori AGE formation in vitro more efﬁciently than PM (Rahbar and Figarola 2003). A lipophilic derivative of vitamin B1, benfotiamine, reduces the accumulation of hexose and triose phosphates by shunting these intermediates to pentose phosphate pathway and improving the activity of transketolase (Stracke et al. 2001). Experimental animal model studies have shown that benfotiamine can reduce diabetic complications like nephropathy and retinopathy (Hammes et al. 2003). Another approach to reduce accumulation of AGE formation is to reverse the process by AGE cross-link breakers such as N-phenacyl thiazolinium bromide and N-phenacyl-4,5-dimethylthiazolium chloride (ALT-711/alagebrium). “RAGE blockers” comprise of agents that trap AGE with soluble RAGE (sRAGE), block RAGE and inhibit signal transduction mediated by AGE–RAGE. Administration of recombinant sRAGE in animal models has shown to suppress atherosclerosis, neuronal dysfunction and diabetes (Park et al. 1998). Novel AGE inhibitors: A link to gold nanoparticles Aldose and ketose sugars were originally thought to be the sole precursors of AGEs. Current research indicates that RCS generated from carbohydrate, lipid and amino acid metabolisms such as MG, glyoxal, 3-DG and malondialdehyde are even more reactive and are potent precursors of AGE formation and protein cross-linking (Bonnefont-Rousselot et al. 2000). Known AGE inhibitors with renoprotective effects such as AG, PM and OPB-9195 are thought to prevent AGE accumulation by interacting with these highly reactive RCS and acting as carbonyl traps, thereby limiting oxidative damage to tissues. AG reacts with α-dicarbonyls such as glyoxal, MG and 3-DG to form triazine derivatives, whereas PM was shown to interact and form adducts with Glyoxal and MG (Lv et al. 2010). It has also been suggested that the chelating activity of AGE inhibitors and AGE breakers at therapeutic concentrations may contribute to their inhibition of AGE formation and protection against development of diabetic complications (Nagai et al. 2012). Most of the novel LR compounds analyzed show greater chelating activities than PM and AG. LR-9, LR-59, LR-74 and LR-90 are potent chelators, with IC50 of 50–275 μM. Spectroscopic 985 S Ahmad et al. studies conﬁrm the formation of inhibitor–copper complexes. The study suggests that metal-catalyzed oxidation plays a critical role in glucose-induced modiﬁcations of the collagen. Figure 4 shows the schematic representation of AGEs, ROS, RNS under hyperglycemic condition and their therapeutic interventions along with their check points. Anti-glycation activity of gold nanoparticles (GNPs) was ﬁrst reported in year 2009 to inhibit the cataract formation as a consequence of the glycation reaction (Singha et al. 2009). The prevention of glycation of α-crystallin was done by conjugation with GNPs. Formation of advanced glycosylic end products is prevented by using bioconjugated GNPs, even if a strong glycating agent such as fructose is used. In addition, the nanoconjugation approach can provide some important information on the structural distribution of any dynamic chaperone protein. Because GNPs are biocompatible, their reported antiglycation activity may have therapeutic medical implications. In another study, the extent of nonenzymatic glycation of HSA was monitored in order to estimate the formation of HSA-related AGEs in the presence of 2 nm GNPs. Physiological concentrations of HSA and glyceraldehyde mixtures, incubated with various concentrations of negatively charged 2 nm GNPs, resulted in a lower reaction rate than mixtures without 2GNP. Moreover, increasing concentrations of GNPs exhibited a pronounced reduction in AGE formation (Seneviratne et al. 2012). Moreover, it has also been suggested that combination of GNP, and antioxidants like Epigallocatechin gallate signiﬁcantly accelerated diabetic cutaneous wound healing through angiogenesis regulation and anti-inﬂammatory effects. This might be due to blockade of RAGE by antioxidant agents and nanoparticles may restore effective wound healing in diabetic ulcer. Furthermore apart from GNPs, silver nanoparticles (Ag-NPs) have also shown to effectively reduce the menace of the glycation reaction. It has been determined the effects of Ag-NPs on AGEs-induced endothelial cell permeability. The AGE-bovine serum albumin (BSA) increased the dextran ﬂux across a PREC monolayer and Ag-NPs blocked the solute ﬂux induced by AGE-BSA. It was also demonstrated that Ag-NPs could inhibit the AGE-BSA-induced permeability via Src kinase pathway. There are reports where researchers have proved that GNPs alone (Seneviratne et al. 2012) and GNPs bioconjugated with protein (Singha et al. 2009) can reduce the rate of nonenzymatic modiﬁcation of proteins responsible for glycation. The role of GNPs in the targeted delivery of drugs is one of the most promising aspects. For the bioconjugation of drug with GNPs a robust and stable protein, albumin was chosen as a capping agent over GNPs. The role of albumin as a drug carrier for last several years has emerged wonderfully because it does not only provide the stability to the drugs but also improves the half-life of drugs/active proteins/peptides. The conjugation was carried out either by simple physical adsorption of the drugs onto GNPs or via the use of alkanethiol linkers (Kratz 2008). Biodistribution homogeneity, penetration via the hematoencephalic barrier and interaction with reticuloendothelial system completely depends on the size of GNPs. The rapid reduction in particle concentration in blood and their prolonged retention in the organism is associated with the functioning of the hepatobiliary system. It takes 3–4 months for the accumulated 986 particles to be excreted from the liver and spleen. Also, available data allow for the reasonable conclusion that colloidal particles with a size of 3–100 nm are not toxic, provided that the threshold dose does not exceed a value of the order of 1012 particles/mL (Dykman and Khlebtsov 2011). A huge number of drugs have been successfully bioconjugated with GNPs and have been approved for clinical trials. The GNPs have also been used in multimodal delivery systems, when a GNP is loaded with several therapeutic agents (both hydrophilic and hydrophobic) and auxiliary agents, such as target molecules and dyes for photodynamic therapy (Kim et al. 2009). There are a large number of antitumor agents and antibiotics have been bioconjugated with GNPs such as paclitaxel (Paciotti et al. 2006), methotrexate (Chen et al. 2004), hemcytabin (Patra et al. 2008), 5-ﬂuorouracil (Agasti et al. 2009), doxorubicin (Asadishad et al. 2010), vancomycin (Gu et al. 2003). The bioconjugation increased stability and efﬁcacy up to 40% of all the drugs used for bioconjugation. Therefore, it is argued that bioconjugation of AGE inhibitors with GNPs will deﬁnitely decrease the Ki substantially and hence doses and ultimately toxicity. The internalization of nanoparticles is taken place by receptor-mediated endocytosis (Chithrani et al. 2006), whereas passive diffusion also plays an active role in the process of internalization. The regulation of internalization of nanoparticles depends upon the interaction of nanoparticles with serum proteins which interact with different nanoparticles based on their hydrophobic properties and impart them equal effective charge irrespective of their initial charge characteristics. The internalization also depend on the size, shape, charge and surface functionalities which interact with cell surface receptors of GNPs (Duncan et al. 2010). In fact, proteins glycated to different extents showed the formation of nanoparticles of different size and eventually their plasmon intensities were also different. This implied that glycated proteins cause some attenuation of the particle formation that led to only smaller nanoparticle formation. One possible example of such negative control on the particle size formation may arise from proximity of glycation-prone sites on which GNPs preferentially form on the protein surface. This as well as other structural studies with nanoparticle protein conjugates, prompted to explore the possibility of prevention of glycation (Bhattacharya et al. 2007). The origin of such resistance against glycation is intriguing. The amino acids containing the free amino group (lysine) are potent sites for glycation in addition to the N-terminal amino acid. On the other hand, GNPs competitively bind to these free amino groups. The observation that the glycation will decrease upon masking of the free amino groups (e.g. those residing on the lysine residues) may be compared with the present observation (Hu et al. 2005). There is a strong implication that the gold seeding process is favored at sites where such free amino groups are present. That amino acid residues such as lysine may be speciﬁc targets (other than thiolcontaining residues like cysteine or methionine) may help in the design of speciﬁc protein targets by GNPs. The search for antiglycating agents is itself an important clinical issue, in that glycation is not conﬁned to diabetic patients, rather glycation leads to damage of several other physiologically important proteins such as collagen, albumin and importantly Hb. Few antiglycating agents have been already reported in the Glycation and its inhibition by nanoconjugation Table I. Bioconjugation of GNPs with different proteins and their association with different metabolic disorders S. no. Conjugation of Au with protein Development of metabolic disorders References 1. 2. 3. 4. 5. 6. 7. 8. α-Crystallin Plasma proteins and amino acids (Au–S or Au–NH2 bonds) Tumor necrosis factor Crystallin protein (masking of protein amine) Collagen Serum albumin Trypsin, bovine serum albumin and transferrin RAGE protein Neurodegenerative diseases Diabetes, cancer Colon cancer Alzheimer’s Aging Diabetes, Alzheimer’s Diabetes, cancer, neurodegenerative diseases Diabetic ulcer, inflammation Singha et al. (2009) Bhattacharya et al. (2007) Rahim et al. (2014) Singha et al. (2009) Kim et al. (2012), Shimasaki et al. (2011) Seneviratne et al. (2012) Rahim et al. (2014) Chen et al. (2012) literature, and apparently a nontoxic agent like gold showing antiglycating properties may itself have important clinical signiﬁcance (Hipkiss 2000; Khan et al. 2013). In the light of the above explanations, this review is aiming to exploit its preventive effect on glycation by reducing the concentrations of the AG or other drugs showing toxicity at higher concentrations but has antiglycation effect. These drugs might be used for nanoconjugation using GNPs, thus reducing the toxic concentration to minimal. The present concern on bioconjugation of AG with GNPs is the need of the hour, as this could overcome the problem of side effects by increasing the efﬁcacy at the reduced doses. To enhance activity of all the above novel drugs at reduced dozes, such as LRs (LR-74, LR-90) and others including AG in bioconjugation with GNPs, might prove to be more accurate and speciﬁc novel inhibitor of DNA, protein and LDL glycation at reduced doses. Table I enlists the bioconjugation of GNPs with different proteins and their association with different metabolic disorders. Future prospects Since very few work has been done on AGEs inhibition using nanoparticles as drug delivery system (Kim et al. 2012; Seneviratne et al. 2012); therefore, it would be interesting to see the inhibition of AGEs using various bioconjugated inhibitors (LR74, LR90 and AG) with GNPs. In the light of above explanations, in general, this study holds strong future prospects with the development of new, more effective and more speciﬁc inhibitors. The inhibitors will be developed based on structure–activity relationship. This study will help to identify the new targets for AGEs. Since there are various diseases associated with glycation and its end products, such as diabetes, cancer, arthritis, atherosclerosis, ageing and neurodegenerative disorders such as Alzheimer’s and Parkinson’s; therefore, targeting a site could result in prevention or protection against these drowning and dreaded diseases. Conclusions There is a considerable body of evidence implicating the formation and accumulation of AGEs as a major factor in the development of diabetic complications, atherosclerosis, Alzheimer’s disease and the normal aging process. The signiﬁcance of this phenomenon becomes more evident where tight association of lipoxidation reactions, overproduction of reactive oxygen species and overgeneration of RCS with the process of AGE formation are considered. Cellular damage by AGEs comes from cross-linking and remodeling of structural proteins such as collagens and metabolic enzymes which affects their physiological functions. Furthermore, tissue damage particularly in vascular endothelial cells may originate by triggering of key cell signaling systems and stimulation of inappropriate cellular activities through secretion of cytokines and vascular cell adhesion molecules. Thus, therapeutic interventions should not only target AGE formation but also AGE-protein cross-link formation. Considering the complexity of pathways and reactions involved in AGE formation, it is not only the end products such as AGE and ALE but also highly reactive carbonyl intermediates responsible for their formation that is toxic to the cells. Therefore, it should be targeted in designing inhibitors that speciﬁcally react with each committed step and intermediate products of important pathways. Another factor to be considered is the fact that glycoxidized proteins generate ROS and induce oxidative stress through the reaction with RAGE which activates and shoot up the oxidative stress thus imbalancing the shift more towards the damaging side. Also, ROS is generated by other reactions in the cascade of AGE formation such as MG and Schiff base pathways leading to lipoxidation and oxidative damage to cells. Therefore, strategies such as suppression of receptor signaling pathways (e.g. RAGE antagonists), and the use of antioxidants and α-oxoaldehyde scavengers will be helpful. Nonetheless, compounds that possess both metal chelating and carbonyl scavenging properties are ideal to inhibit both oxidative and carbonyl stresses. Such class of compounds may also be effectors of AGE receptors such as RAGE that is involved in cell signaling pathways. Unfortunately, from a large number of naturally occurring and synthetic compounds reported previously as AGE inhibitors, only the mechanism of action of a few compounds have been studied extensively to date. Unraveling the mechanism(s) of action of these inhibitors is essential for understanding the roles of AGE in the pathogenesis of a number of age-related chronic diseases and to design more effective therapeutic strategies for these diseases in the future. Last but not the least, bioconjugated LR-74, LR-90 and AG might prove to be novel inhibitor for glycation of DNA, proteins and LDL as very few studies has been performed till date to stop/inhibit AGE formation using gold as a nanoparticle. This kind of inhibition will be more accurate and precise with reduced doses. 987 S Ahmad et al. Abbreviations AG, aminoguanidine; AGEs, advanced glycation end products; Ag-NPs, silver nanoparticles; ALEs, advanced lipoxidation end products; apo B, apolipoprotein B; BSA, bovine serum albumin; CEdG, carboxy ethyl deoxygunaosine; CEL, carboxyethyl lysine; CML, carboxymethyl lysine; DAG, diacyl glycerol generation; DNA-AGEs, DNA advanced glycation end products; GNPs, gold nanoparticles; HSA, human serum albumin; HEC, human endothelial cell; LDLs, low-density lipoproteins; LR, Lalezari-Rahbar; MG, methylglyoxal; PM, pyridoxamine; RAGE, receptor for AGE; RCS, reactive carbonyl species; ROS, reactive oxygen species; sRAGE, soluble RAGE Acknowledgements The authors are highly thankful to the Vice Chancellor of the Integral University, Prof. Waseem Akhtar Sb. for the infrastructure support provided to the Department of Bio-Sciences. Conﬂict of interest statement None declared. 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