**2. The ageing process**

The process of aging is accepted as an inevitable normal part of the life cycle of each and every living organism. Aging can be grossly defined as an overall decline in biological functions. Thus, aging involves gradual changes in the body such as reduced immunity, loss of muscle strength, stiffening of the arterial wall, loss of elasticity and wrinkling of the skin, and decline in memory, all of which result in increasing weakness, risk of developing diseases, and ultimately death. These changes take place at the cellular, organ and the whole organism level. The whole process of aging unfolds very clearly in species with a long life span such as human beings. Cellular aging ultimately translates into whole body aging.

Hayflick et al. [1] first described cellular senescence in the sixties when they showed that normal cells had a limited ability to proliferate in culture. Cellular senescence is believed to be initiated by increased cellular stress [2, 3]. Factors contributing to cellular stress and aging include dysfunctional telomeres (telomere length) [4, 5], DNA damage [6] and mitogenic or oncogenic stimuli and signals [2, 4, 5]. The factors such as age and oxidative

Aging: Drugs to Eliminate Methylglyoxal, a Reactive

explained in reviews by Haliwell [20, 21].

could lead to accelerated aging [7, 9, 10].

lower rate of production of oxygen radicals [34].

unequivocal support for the free radical theory of aging [40].

Glucose Metabolite, and Advanced Glycation Endproducts 683

and prevent the generation of oxidative stress resulting from an excess of free radicals [17- 22]. The formation of free radicals and the function of antioxidants have been nicely

The free radical theory of aging was proposed by Denham Harman in 1956 [23]. The free radical theory of aging attributes the aging process to cumulative cellular damage inflicted by the reaction of free radicals with key functional cellular and tissue constituents resulting in impaired function, disease and death [23]. The discovery of an antioxidant enzyme, superoxide dismutase (SOD) [24], which plays a key role to eliminate superoxide anion levels, provided some validity to the free radical theory, which was not initially accepted by many. The mitochondrial respiratory chain is a major source of free radicals, mainly in the form of superoxide anions, which cause damage to the mitochondria and reduce life span [25, 26]. The damage inflicted by ROS, especially to DNA [7], rather than the metabolic rate, showed a greater correlation with life span [8]. Damage to DNA was formulated into the somatic mutation theory, which states that genetic mutations caused by an excess of free radicals

The fact that increased production of ROS in the mitochondria can reduce life span was supported by several studies. Thus, Ku *et al*., [27] showed that the rates of mitochondrial superoxide anion and hydrogen peroxide generation were inversely correlated to maximum life span potential when they compared seven different mammalian species with different life spans ranging from 3.5 to 30 years. Similarly, ROS production was higher in heart mitochondria of the rat, which has a life span of about 4 yrs, than in the long-lived pigeon, which has a longer life span of 35 yrs [28]. Theoretically, therefore, if the free radical production is diminished, the life span should increase. This has been demonstrated in several species. Thus, over expression of SOD and catalase in the worm *Caenorhabditis elegans* (*C. Elegans*) through *age-1* alleles, increases their oxidative defenses and life span by 65% longer on average [29, 30]. Increased activity of SOD and reduced oxidative stress in the transgenic *Drosophila* (*Drosophila melanogaster*) flies also slows the aging process and results in a longer life [31, 32]. Also, over expression of catalase in the peroxisome, the mitochondria or the nucleus in transgenic mice, reduced oxidative damage, hydrogen peroxide production, and delayed the development of cardiac pathology and cataract formation along with an average increase of 5.5 months in the life span [33]. The observation that longlived animals have lower levels of antioxidant enzymes was explained as being due to a

Interestingly, some of the studies in rodents did not produce the expected results. For example, the administration of antioxidants [35], or over expression of CuZn SOD and catalase in mice [36], or SOD in rats [37], did not increase their life spans. In rodents, one reason for the lack of additional protective effects, which are normally associated with an increase in antioxidants, could be their ability to synthesize vitamin C [38], which might already be providing the required protection. This was verified by knocking out the vitamin C synthesizing enzyme, L-gluconolactone oxidase (GLO) in mice, which then have to depend on dietary vitamin C [39]. GLO knockout mice had damaged aortic walls when they were fed a diet low in vitamin C, which underlined the importance of the constitutive antioxidant function of vitamin C in rodents [39]. Thus, studies in rodents do not provide

stress affect telomere length and telomerase activity which in turn affects cellular senescence [4]. Oxidative stress has been shown to damage DNA and affect life span [7-10]. A controversial view of cellular senescence is that it is an important protective mechanism against transformation of the cell into a malignant phenotype, in which case it would affect only mitotically active cells [2, 3]. The molecular mechanisms involved in cellular senescence are still being unraveled and will not be considered further in this review. The focus of this review will be on MG, a reactive dicarbonyl metabolic intermediate produced in the body, AGEs, and oxidative stress, all of which are interrelated and affect cellular as well as whole body aging. We will discuss some compounds that can scavenge MG, prevent the formation of AGEs (inhibitors) or break the existing AGEs (AGE breakers).

#### **3. Theories of aging**

Aging has been attributed to a number of different causes which have been presented in the form of different theories. These theories are based broadly on two different ideas, one of which is programmed life processes (program theories, e.g. Biological Clock theory, Limited Number of Proliferation theory), and the other one is of errors, mainly at the DNA and gene level, in life processes (error theories, e.g. Disease theory, Cross-linking theory, Rate of living theory, Free radical theory). A number of theories of aging are based on the combination of these two ideas, i.e. program theories and error theories [11-14].

Changes at the cellular level ultimately affect the whole body. The cell is a dynamic centre of ongoing metabolic activity driven by almost constant use of oxygen. Reasonably, the metabolic activity may affect survival or the death of the cell. The 'Rate of living theory' implicates the role of metabolism in aging, which is based on the observation that animals with higher metabolic rates often have shorter life spans. Since the metabolic processes and oxygen consumption can also generate oxidative stress, an excess of which is deleterious for the cell, the 'Free radical theory' of aging has become one of the more popular theories. The free radical theory proposes a connection between the metabolic rate and aging through an increased oxidative stress generation.

#### **4. Free radical theory of aging**

Max Rubner proposed the 'rate of living theory' early in the 20th century [15]. He observed that larger animals, which generally have slower metabolic rates, live longer than smaller animals with faster metabolic rates [15]. Even though it is now common knowledge that metabolism is associated with the generation of free radicals, it was Commoner *et al*. [16] who discovered the formation of free radicals *in vivo*. Commoner et al. [16] found that an increase in an organism's metabolic activity can increase the concentration of endogenous free radicals. Free radicals are atoms or molecules with an unpaired electron in an orbit, making them highly reactive. The high reactivity of free radicals makes them deleterious for cells because they react with proteins, lipids, DNA and other biomolecules, and disrupt their structure and function. Free radicals can be derived from oxygen mainly in the form of superoxide anions (O2•-) and hydroxyl radicals (·OH), which are known as reactive oxygen species (ROS). Free radicals can also be in the form of highly reactive non-radicals which do not have an unpaired electron in their orbit, such as hydrogen peroxide (H2O2). Normally, the cells and the body have adequate antioxidant defenses which can neutralize free radicals

stress affect telomere length and telomerase activity which in turn affects cellular senescence [4]. Oxidative stress has been shown to damage DNA and affect life span [7-10]. A controversial view of cellular senescence is that it is an important protective mechanism against transformation of the cell into a malignant phenotype, in which case it would affect only mitotically active cells [2, 3]. The molecular mechanisms involved in cellular senescence are still being unraveled and will not be considered further in this review. The focus of this review will be on MG, a reactive dicarbonyl metabolic intermediate produced in the body, AGEs, and oxidative stress, all of which are interrelated and affect cellular as well as whole body aging. We will discuss some compounds that can scavenge MG, prevent the formation

Aging has been attributed to a number of different causes which have been presented in the form of different theories. These theories are based broadly on two different ideas, one of which is programmed life processes (program theories, e.g. Biological Clock theory, Limited Number of Proliferation theory), and the other one is of errors, mainly at the DNA and gene level, in life processes (error theories, e.g. Disease theory, Cross-linking theory, Rate of living theory, Free radical theory). A number of theories of aging are based on the

Changes at the cellular level ultimately affect the whole body. The cell is a dynamic centre of ongoing metabolic activity driven by almost constant use of oxygen. Reasonably, the metabolic activity may affect survival or the death of the cell. The 'Rate of living theory' implicates the role of metabolism in aging, which is based on the observation that animals with higher metabolic rates often have shorter life spans. Since the metabolic processes and oxygen consumption can also generate oxidative stress, an excess of which is deleterious for the cell, the 'Free radical theory' of aging has become one of the more popular theories. The free radical theory proposes a connection between the metabolic rate and aging through an

Max Rubner proposed the 'rate of living theory' early in the 20th century [15]. He observed that larger animals, which generally have slower metabolic rates, live longer than smaller animals with faster metabolic rates [15]. Even though it is now common knowledge that metabolism is associated with the generation of free radicals, it was Commoner *et al*. [16] who discovered the formation of free radicals *in vivo*. Commoner et al. [16] found that an increase in an organism's metabolic activity can increase the concentration of endogenous free radicals. Free radicals are atoms or molecules with an unpaired electron in an orbit, making them highly reactive. The high reactivity of free radicals makes them deleterious for cells because they react with proteins, lipids, DNA and other biomolecules, and disrupt their structure and function. Free radicals can be derived from oxygen mainly in the form of superoxide anions (O2•-) and hydroxyl radicals (·OH), which are known as reactive oxygen species (ROS). Free radicals can also be in the form of highly reactive non-radicals which do not have an unpaired electron in their orbit, such as hydrogen peroxide (H2O2). Normally, the cells and the body have adequate antioxidant defenses which can neutralize free radicals

combination of these two ideas, i.e. program theories and error theories [11-14].

of AGEs (inhibitors) or break the existing AGEs (AGE breakers).

**3. Theories of aging** 

increased oxidative stress generation.

**4. Free radical theory of aging** 

and prevent the generation of oxidative stress resulting from an excess of free radicals [17- 22]. The formation of free radicals and the function of antioxidants have been nicely explained in reviews by Haliwell [20, 21].

The free radical theory of aging was proposed by Denham Harman in 1956 [23]. The free radical theory of aging attributes the aging process to cumulative cellular damage inflicted by the reaction of free radicals with key functional cellular and tissue constituents resulting in impaired function, disease and death [23]. The discovery of an antioxidant enzyme, superoxide dismutase (SOD) [24], which plays a key role to eliminate superoxide anion levels, provided some validity to the free radical theory, which was not initially accepted by many.

The mitochondrial respiratory chain is a major source of free radicals, mainly in the form of superoxide anions, which cause damage to the mitochondria and reduce life span [25, 26]. The damage inflicted by ROS, especially to DNA [7], rather than the metabolic rate, showed a greater correlation with life span [8]. Damage to DNA was formulated into the somatic mutation theory, which states that genetic mutations caused by an excess of free radicals could lead to accelerated aging [7, 9, 10].

The fact that increased production of ROS in the mitochondria can reduce life span was supported by several studies. Thus, Ku *et al*., [27] showed that the rates of mitochondrial superoxide anion and hydrogen peroxide generation were inversely correlated to maximum life span potential when they compared seven different mammalian species with different life spans ranging from 3.5 to 30 years. Similarly, ROS production was higher in heart mitochondria of the rat, which has a life span of about 4 yrs, than in the long-lived pigeon, which has a longer life span of 35 yrs [28]. Theoretically, therefore, if the free radical production is diminished, the life span should increase. This has been demonstrated in several species. Thus, over expression of SOD and catalase in the worm *Caenorhabditis elegans* (*C. Elegans*) through *age-1* alleles, increases their oxidative defenses and life span by 65% longer on average [29, 30]. Increased activity of SOD and reduced oxidative stress in the transgenic *Drosophila* (*Drosophila melanogaster*) flies also slows the aging process and results in a longer life [31, 32]. Also, over expression of catalase in the peroxisome, the mitochondria or the nucleus in transgenic mice, reduced oxidative damage, hydrogen peroxide production, and delayed the development of cardiac pathology and cataract formation along with an average increase of 5.5 months in the life span [33]. The observation that longlived animals have lower levels of antioxidant enzymes was explained as being due to a lower rate of production of oxygen radicals [34].

Interestingly, some of the studies in rodents did not produce the expected results. For example, the administration of antioxidants [35], or over expression of CuZn SOD and catalase in mice [36], or SOD in rats [37], did not increase their life spans. In rodents, one reason for the lack of additional protective effects, which are normally associated with an increase in antioxidants, could be their ability to synthesize vitamin C [38], which might already be providing the required protection. This was verified by knocking out the vitamin C synthesizing enzyme, L-gluconolactone oxidase (GLO) in mice, which then have to depend on dietary vitamin C [39]. GLO knockout mice had damaged aortic walls when they were fed a diet low in vitamin C, which underlined the importance of the constitutive antioxidant function of vitamin C in rodents [39]. Thus, studies in rodents do not provide unequivocal support for the free radical theory of aging [40].

Aging: Drugs to Eliminate Methylglyoxal, a Reactive

**Pyruvate**

**Fatty acid Acetone,** 

**Sugar / glucose**

**AGEs**

MG [60-62].

**Krebs cycle**

**Triacylglycerol**

Glucose Metabolite, and Advanced Glycation Endproducts 685

(lactoylglutathione lyase) and glyoxalase II (hydroxyacylglutathione hydrolase) [57-59] (Fig. 2). Reduced glutathione (GSH) plays a key role by binding MG and presenting it to glyoxalase I. Thus, adequate availability of GSH is important in keeping MG levels low in the body. For this reason enzymes involved in the synthesis and recycling of GSH, such as glutathione peroxidase and glutathione reductase are also important in the metabolism of

> *Polyol pathway*

> > **Fructose**

**Sorbitol**

**Glucose**

**F-1,6-di-P**

**+ Proteins**

*AMO*

**Glycerol**

**acetol**

**DHAP G-3-P**

Fig. 2. A schematic of key sources and steps of methylglyoxal (MG) formation from

intermediates of glucose, protein and fat metabolism, and its degradation by the glyoxalase enzymes. Abbreviations: AGEs – advanced glycation endproducts; AMO – amine oxidase; DHAP – dihydroxacetone phosphate; FA – fatty acid; F-1-P – fructose-1-phosphate; F-1,6-di-P – fructose-1,6-diphosphate; F-6-P – fructose-6-phosphate; G-3-P – glyceraldehyde-3 phosphate; G-6-P – glucose-6-phosphate; ROS – reactive oxygen species; SSAO – semicarbazide-sensitive acetone/acetol mono-oxygenase; GSH, reduced glutathione.

Despite the efficient glyoxalase system, MG levels can increase significantly in the plasma and different organs such as the aorta and the kidneys [61, 63-66]. We have shown that MG levels are elevated in the plasma, aorta and kidney of fructose-fed Sprague-Dawley rats and spontaneously hypertensive rats (SHR) [61, 63-65]. Patients with type 1 and type 2 diabetes have 2-6 fold higher plasma levels of MG compared to healthy people [67, 68]. MG possibly plays a role in the pathogenesis of insulin resistance and type 2 diabetes as shown by several *in vitro* [69-71], and by our recent *in vivo* study in acute [66] and chronic MG-treated

**Free radicals ROS**

**METHYLGLYOXAL**

*SSAO*

**S,D-lactoyl glutathione** *Glyoxalase I*

*Glyoxalase II*

**Hemithioacetal**

**AGING D-lactic acid**

*+ GSH*

**Protein**

**Glycine, threonine**

**Aminoacetone**

Another way of increasing free radical production and oxidative stress is by increasing total caloric intake, which can be easily done be feeding an excess of glucose. A correlation between life span and dietary caloric intake was reported in rats and mice by McCay *et al*. [41]. One quantitative estimate was provided in the study by Weindruch and Walford [42] who showed that a 40% reduction in dietary caloric intake extended maximum life span by one third. A high dietary caloric intake causes an increased rate of DNA damage [43], due to a high metabolic rate which in turn results in higher amounts of superoxide anion, hydrogen peroxide and hydroxyl radical formation [44].

#### **5. Methylglyoxal**

Chemically, MG, or pyruvaldehyde, is a highly reactive electrophilic α,β-dicarbonyl compound [45, 46] (Fig. 1). MG has been proposed to be formed mainly during glycolysis, through spontaneous nonenzymatic transformation of triose phosphates [45, 47-49] (Fig. 2). MG synthase has been proposed to convert the triose phosphate intermediate, dihydroxyacetone phosphate (DHAP), into MG, especially when inadequate inorganic phosphate is available [50, 51]. Other sources of MG, which are believed to produce lower amounts of MG, include intermediates of protein and fatty acid metabolism, such as aminoacetone produced from L-threonine and glycine [52, 53], and acetone [54, 55], respectively (Fig. 2). Semicarbazide-sensitive amine oxidase (SSAO) catalyzes the breakdown of aminoacetone [52, 55, 56], while acetone and acetol mono-oxygenase (AMO) converts acetone to acetol and acetol to MG, respectively [54] (Fig. 2). SSAO is found in substantial amounts in the vascular smooth muscle cells and the plasma [55].

N-phenacyl-4,5-dimethyl-1,3-thiazolium chloride Alagebrium chloride (ALT-711)

Metformin (dimethylbiguanide)

Fig. 1. Structure of methylglyoxal (MG) and three compounds with an ability to bind MG or inhibit the formation of advanced glycation endproducts (AGEs) or break formed AGEs. These compounds are discussed in this review.

After MG is formed, it is rapidly degraded to D-lactic acid by the highly efficient and ubiquitous glyoxalase system, which consists of two key enzymes, glyoxalase I

Another way of increasing free radical production and oxidative stress is by increasing total caloric intake, which can be easily done be feeding an excess of glucose. A correlation between life span and dietary caloric intake was reported in rats and mice by McCay *et al*. [41]. One quantitative estimate was provided in the study by Weindruch and Walford [42] who showed that a 40% reduction in dietary caloric intake extended maximum life span by one third. A high dietary caloric intake causes an increased rate of DNA damage [43], due to a high metabolic rate which in turn results in higher amounts of superoxide anion,

Chemically, MG, or pyruvaldehyde, is a highly reactive electrophilic α,β-dicarbonyl compound [45, 46] (Fig. 1). MG has been proposed to be formed mainly during glycolysis, through spontaneous nonenzymatic transformation of triose phosphates [45, 47-49] (Fig. 2). MG synthase has been proposed to convert the triose phosphate intermediate, dihydroxyacetone phosphate (DHAP), into MG, especially when inadequate inorganic phosphate is available [50, 51]. Other sources of MG, which are believed to produce lower amounts of MG, include intermediates of protein and fatty acid metabolism, such as aminoacetone produced from L-threonine and glycine [52, 53], and acetone [54, 55], respectively (Fig. 2). Semicarbazide-sensitive amine oxidase (SSAO) catalyzes the breakdown of aminoacetone [52, 55, 56], while acetone and acetol mono-oxygenase (AMO) converts acetone to acetol and acetol to MG, respectively [54] (Fig. 2). SSAO is found in

> C C N H H2N N

CH3

CH3

Metformin (dimethylbiguanide)

NH

NH

C N H H2N NH2

NH

Aminoguanidine

substantial amounts in the vascular smooth muscle cells and the plasma [55].

hydrogen peroxide and hydroxyl radical formation [44].

O

H

+N

Methylglyoxal

O

C

H3C O

C

Cl -

N-phenacyl-4,5-dimethyl-1,3-thiazolium chloride Alagebrium chloride (ALT-711)

These compounds are discussed in this review.

CH3

S

CH3

Fig. 1. Structure of methylglyoxal (MG) and three compounds with an ability to bind MG or inhibit the formation of advanced glycation endproducts (AGEs) or break formed AGEs.

After MG is formed, it is rapidly degraded to D-lactic acid by the highly efficient and ubiquitous glyoxalase system, which consists of two key enzymes, glyoxalase I

**5. Methylglyoxal** 

(lactoylglutathione lyase) and glyoxalase II (hydroxyacylglutathione hydrolase) [57-59] (Fig. 2). Reduced glutathione (GSH) plays a key role by binding MG and presenting it to glyoxalase I. Thus, adequate availability of GSH is important in keeping MG levels low in the body. For this reason enzymes involved in the synthesis and recycling of GSH, such as glutathione peroxidase and glutathione reductase are also important in the metabolism of MG [60-62].

Fig. 2. A schematic of key sources and steps of methylglyoxal (MG) formation from intermediates of glucose, protein and fat metabolism, and its degradation by the glyoxalase enzymes. Abbreviations: AGEs – advanced glycation endproducts; AMO – amine oxidase; DHAP – dihydroxacetone phosphate; FA – fatty acid; F-1-P – fructose-1-phosphate; F-1,6-di-P – fructose-1,6-diphosphate; F-6-P – fructose-6-phosphate; G-3-P – glyceraldehyde-3 phosphate; G-6-P – glucose-6-phosphate; ROS – reactive oxygen species; SSAO – semicarbazide-sensitive acetone/acetol mono-oxygenase; GSH, reduced glutathione.

Despite the efficient glyoxalase system, MG levels can increase significantly in the plasma and different organs such as the aorta and the kidneys [61, 63-66]. We have shown that MG levels are elevated in the plasma, aorta and kidney of fructose-fed Sprague-Dawley rats and spontaneously hypertensive rats (SHR) [61, 63-65]. Patients with type 1 and type 2 diabetes have 2-6 fold higher plasma levels of MG compared to healthy people [67, 68]. MG possibly plays a role in the pathogenesis of insulin resistance and type 2 diabetes as shown by several *in vitro* [69-71], and by our recent *in vivo* study in acute [66] and chronic MG-treated

Aging: Drugs to Eliminate Methylglyoxal, a Reactive

**7. Methylglyoxal, AGEs, oxidative stress and aging** 

AGEs formation. AGEs also induce oxidative stress.

**8. Methylglyoxal and oxidative stress** 

neuroblastoma cells [104].

PPAR-α [104].

(Fig. 3).

[112].

proteins in cells [105].

in tissues [93].

Glucose Metabolite, and Advanced Glycation Endproducts 687

imidazolones [91, 92]. The presence of these AGEs can be detected immunohistochemically

The damage inflicted by oxidative stress and the formation of intracellular AGEs likely contribute to cellular aging. From this point of view, both increased MG and AGEs would cause accelerated cellular aging. MG would be a double-edged sword because it is a potent inducer of oxidative stress [17, 62, 94, 95], as discussed below, and it is a major precursor of

The role of MG in inducing oxidative stress is well established [17]. Several studies have helped to develop an integrated view of the multiple pathways activated by MG to increase oxidative stress (Fig. 3). The reader is referred to our earlier review on MG and oxidative stress [17]. MG increases the formation of superoxide [94, 96-99], hydrogen peroxide and peroxynitrite [94, 95, 98, 100], proinflammatory cytokines, such as interleukin 1β (IL-1β) [101], interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) [67, 101], in different cell types such as VSMCs [62, 94, 95], endothelial cells [102], rat kidney mesangial cells [97], rat hepatocytes [100], neutrophils [67, 98], platelets [99], cultured neural cells from rat hippocampus [101], cultured cortical neurons [103], and SH-SY5Y

MG has been shown to increase the activity of several prooxidant enzymes such as NADPH oxidase [94, 97] (Fig. 3), p38 MAPK [98, 102], and increase the of expression of JNK and

Excess superoxide can react with nitric oxide (NO) to form peroxynitrite (ONOO-) [105] (Fig. 3). Peroxynitrite is a strong oxidant and nitrating agent. Because of its oxidizing properties, peroxynitrite can damage a wide range of molecules including DNA and

Besides directly increasing free radical production, MG can increase oxidative stress by reducing antioxidants (Fig. 3) such as GSH [104, 106, 107], glutathione peroxidase [108], glutathione reductase [60, 62, 108, 109], and manganese superoxide dismutase (MnSOD) [96], in different cells such as erythrocytes [106, 107], VSMCs [62, 96], and endothelial cells [109]. Reduced antioxidants in turn impair the detoxification of MG, increase its half-life and set up a vicious cycle to cause further oxidant damage. Glutathione peroxidase removes hydrogen peroxide with the help of GSH which in turn is converted to oxidized glutathione (GSSG). Glutathione reductase recycles GSSG to GSH [62, 110]

An increased production of ROS was also observed in monocytes treated with MGmodified albumin [111]. Thus, MG induced thrombosis and inflammation by activating monocytes, induced apoptosis of neutrophils, and caused platelet-neutrophil aggregates

Sprague-Dawley rats [72]. Elevated MG levels are linked to the development of microvascular complications of diabetes such as retinopathy and nephropathy, and other conditions such as atherosclerosis and neurodegenerative diseases [73-77]. MG levels are high in the cerebrospinal fluid of patients with Alzheimer's disease [76].

#### **6. Advanced glycation endproducts**

Unwanted chemical modification of physiologic constituent molecules of the body, which leads to the formation of harmful chemical entities, seems to be an unavoidable part of metabolic processes of the body. One type of modification, known as glycation, a nonenzymatic reaction, is a serious hazard of excess glucose availability in the body. The chemical interaction leading to the formation of AGEs starts when a reducing sugar condenses with the amino groups of proteins at their N terminus or on lysyl side chains (εamino groups) [78]. This nonenzymatic glycation involves a series of post-translational modifications. Glycation begins with the aldehyde or the ketone carbonyl group of the sugar combining with the protein to form an unstable aldimine intermediate or a Schiff base. Later on the Schiff base undergoes an Amadori rearrangement to form a stable Amadori product, a l-amino-l-deoxyfructose derivative with a stable ketoamine linkage, which can get cyclized to form a ring structure [78-80]. The Amadori product can undergo oxidation, degradation or rearrangement and form AGEs, a heterogenous group of products. Auto oxidation of glucose (Wolff pathway) [81] or of the Schiff bases (Namiki pathway) [82] can lead to formation of reactive diacrbonyls, but these pathways which are readily observed at high glucose concentrations *in vitro*, are not predominant *in vivo* [83]. The Maillard reaction, also known as the "browning reaction", involves oxidation of the glycated product which forms a brown coloured product. Glucose, fructose and glucose-6-phosphate are all involved in glycation, albeit at different rates of reaction with glucose, the most important contributor, reacting at a comparatively slower rate than the other two [6]. Increased glucose levels, as seen in diabetic patients, causes more AGEs formation than in healthy people. These AGEs affect the normal function of several proteins and enzymes, and are responsible for aging [74, 80] and the complications of diabetes such as nephropathy and retinopathy [79]. Another way by which the glycation reaction causes damage is through the formation of reactive α-dicarbonyl compounds, such as MG, glyoxal and 3-deoxyglucosone (3-DG), when the sugar molecule undergoes fragmentation [78].

MG can also cause AGEs formation [78, 79]. In fact, MG and two other dicarbonyl metabolic intermediates, 3-DG and glyoxal, are believed to be major sources of intracellular and plasma AGEs formation [79, 84, 85], which are commonly implicated in the aging process. Any MG which is not degraded by the glyoxalase system or aldose reductase, reacts nonenzymatically with arginine or lysine residues of proteins [45] to form irreversible AGEs. This glycation is not random, but it depends on the structural configuration and (or) physical locations of the target proteins [86, 87]. The AGEs produced by the reaction between MG and arginine are hydroimidazolone Nε-(5-hydro-5-methyl-4-imidazolon-2-yl) ornithine and argpyrimidine [88], whereas the AGE, Nε-carboxyethyllysine (CEL) [89, 90] is formed when MG reacts with lysine. Further crosslinking of these AGEs produces fluorescent products such as pentosidine and cross-line, and non-fluorescent ones such as argpyrimidine, methylglyoxal-lysine dimer (MOLD), glyoxal-lysine dimer (GOLD) and

Sprague-Dawley rats [72]. Elevated MG levels are linked to the development of microvascular complications of diabetes such as retinopathy and nephropathy, and other conditions such as atherosclerosis and neurodegenerative diseases [73-77]. MG levels are

Unwanted chemical modification of physiologic constituent molecules of the body, which leads to the formation of harmful chemical entities, seems to be an unavoidable part of metabolic processes of the body. One type of modification, known as glycation, a nonenzymatic reaction, is a serious hazard of excess glucose availability in the body. The chemical interaction leading to the formation of AGEs starts when a reducing sugar condenses with the amino groups of proteins at their N terminus or on lysyl side chains (εamino groups) [78]. This nonenzymatic glycation involves a series of post-translational modifications. Glycation begins with the aldehyde or the ketone carbonyl group of the sugar combining with the protein to form an unstable aldimine intermediate or a Schiff base. Later on the Schiff base undergoes an Amadori rearrangement to form a stable Amadori product, a l-amino-l-deoxyfructose derivative with a stable ketoamine linkage, which can get cyclized to form a ring structure [78-80]. The Amadori product can undergo oxidation, degradation or rearrangement and form AGEs, a heterogenous group of products. Auto oxidation of glucose (Wolff pathway) [81] or of the Schiff bases (Namiki pathway) [82] can lead to formation of reactive diacrbonyls, but these pathways which are readily observed at high glucose concentrations *in vitro*, are not predominant *in vivo* [83]. The Maillard reaction, also known as the "browning reaction", involves oxidation of the glycated product which forms a brown coloured product. Glucose, fructose and glucose-6-phosphate are all involved in glycation, albeit at different rates of reaction with glucose, the most important contributor, reacting at a comparatively slower rate than the other two [6]. Increased glucose levels, as seen in diabetic patients, causes more AGEs formation than in healthy people. These AGEs affect the normal function of several proteins and enzymes, and are responsible for aging [74, 80] and the complications of diabetes such as nephropathy and retinopathy [79]. Another way by which the glycation reaction causes damage is through the formation of reactive α-dicarbonyl compounds, such as MG, glyoxal and 3-deoxyglucosone (3-DG), when

MG can also cause AGEs formation [78, 79]. In fact, MG and two other dicarbonyl metabolic intermediates, 3-DG and glyoxal, are believed to be major sources of intracellular and plasma AGEs formation [79, 84, 85], which are commonly implicated in the aging process. Any MG which is not degraded by the glyoxalase system or aldose reductase, reacts nonenzymatically with arginine or lysine residues of proteins [45] to form irreversible AGEs. This glycation is not random, but it depends on the structural configuration and (or) physical locations of the target proteins [86, 87]. The AGEs produced by the reaction between MG and arginine are hydroimidazolone Nε-(5-hydro-5-methyl-4-imidazolon-2-yl) ornithine and argpyrimidine [88], whereas the AGE, Nε-carboxyethyllysine (CEL) [89, 90] is formed when MG reacts with lysine. Further crosslinking of these AGEs produces fluorescent products such as pentosidine and cross-line, and non-fluorescent ones such as argpyrimidine, methylglyoxal-lysine dimer (MOLD), glyoxal-lysine dimer (GOLD) and

high in the cerebrospinal fluid of patients with Alzheimer's disease [76].

**6. Advanced glycation endproducts** 

the sugar molecule undergoes fragmentation [78].

imidazolones [91, 92]. The presence of these AGEs can be detected immunohistochemically in tissues [93].
