**3.** *In vivo* **AGE generation pathways**

To date, AGEs have been widely studied because of the close involvement in diabetic complications. HbA1c is currently used as a diagnostic criterion and indicator of mean blood glucose levels over a period of 1–2 months in patients with diabetes. Albumin, another representative protein in the blood, is also related to diabetic complications. In patients with diabetes, albumin has been shown to glycate four lysine residues (K199, K281, K439, and K525) in the molecule [21]. In addition, albumin is more easily saccharified than haemoglobin, and its reaction is rapid; thus, blood GA levels fluctuate more than HbA1c levels. Accordingly, gluco-albumin, which has a short half-life, was recently reported as an index of the average blood glucose level over a period of approximately 2 weeks [22].

At the experimental level, bovine serum albumin (BSA) has been used to evaluate the functions of AGEs *in vivo*. Various specific antibodies have been produced by immunisation with glycated AGE-BSA as antigens. Many commercially available AGEs are produced *in vitro* by incubating BSA and d-glucose at 37°C for 8 weeks in 0.2 M phosphate buffer (pH 7.4) and 5 mM DTPA. Farboud et al. reacted BSA with glycolaldehyde to produce pentosidine-BSA and obtained antibodies that recognise CML and pentosidine from this antigen [23]. Takeuchi named these six types of AGEs as glucose-derived AGE-1 (Glc-AGE), glyceraldehyde-derived AGE-2 (Glycer-AGE), glycol aldehyde-derived AGE-3 (Glycol-AGE), methylglyoxal-derived AGE-4 (MGO-AGE), glyoxal AGE-5 (GO-AGE), and 3-deoxyglucosone-derived AGE-6 (3DG-AGE); they then produced specific antibodies against each of the six types [24–26] (**Figure 3**). Using these antibodies, Takeuchi et al. clarified that AGE-2 derived from glyceraldehyde and AGE-3 derived from glycolaldehyde, produced by Schiff bases and Amadori compounds, were closely related to the onset and progression of diabetic retinopathy and nephropathy compared with AGE-1 [27–30]. The authors also demonstrated that these highly toxic AGE-2 and AGE-3 act via receptors for AGEs (RAGE) and therefore named these molecules toxic AGEs (TAGEs) [31], and identified nontoxic AGEs, including AGEs such as CML, pentocidin, and pyrrolin that are generated from glucose and by active trapping and detoxification of highly chemically reactive aldehyde/carbonyl compounds occurring in the body. TAGEs derived from glyceraldehyde, glycolaldehyde, and acetaldehyde are critical to the development and progression of various diseases and should be considered separately from other AGEs [32].

During the production of TAGEs, unique glucose metabolism pathways have been identified in the hyperglycaemic environment associated with diabetes. For example, in the hyperglycaemic environment observed in patients with type 2 diabetes, intracellular glucose levels are abnormally elevated in cells that take up insulin-independent glucose, such as the liver, brain, and placenta. The liver expresses the glucose transporter (GLUT) named as GLUT2, which has a low affinity for and takes up a large amount of glucose. GLUT3, which has a high affinity for glucose, also functions in glucose transport [33]. In such cells, the extra glucose is shunted into the polyol pathway by saturation of the normal glycolytic pathway [34, 35]. The polyol pathway is a side pathway that is activated when glycolysis is stagnant. First, excess glucose, which is not metabolised by glycolysis, is converted to sorbitol (polyol) by aldose reductase, after which sorbitol is metabolised to fructose by sorbitol dehydrogenase. When aldose reductase is enhanced, excessive consumption of its coenzyme NADPH causes a decrease in reduced glutathione and abnormalities in the active oxygen scavenging system. Such an increase in aldose

*Advanced Glycation End Products and Oxidative Stress in a Hyperglycaemic Environment DOI: http://dx.doi.org/10.5772/intechopen.97234*

#### **Figure 3.**

*AGE generation process in vivo. In the living body, AGEs are produced via dicarbonyl compounds generated during glucose metabolism of reducing sugars, such as glucose. In a hyperglycaemic environment, when glycolysis is stopped, the polyol circuit is enhanced, and glyceraldehyde-AGEs are produced. GO-AGEs, glyoxal (GO) derived AGEs; glycol-AGEs, glycolaldehyde-derived AGEs; Glc-AGEs, glucose-derived AGEs; 3-DG-AGEs, 3-deoxyglucosone (3-DG)-derived AGEs; MGO-AGEs, methylglyoxal (MGO)-derived AGEs; glycer-AGEs, glyceraldehyde-derived AGEs; CML, Nε-(carboxymethyl) lysine. This figure has been modified based on the reference [25, 26].*

reductase in type 2 diabetes is thought to worsen haemodynamics and lead to diabetic neuropathy (DN) [36]. Therefore, in patients with diabetes, the concentration of fructose produced from glucose is increased intracellularly because of enhancement of the polyol pathway [37, 38].

Fructose produced by this polyol pathway is thought to have a stronger protein glycation ability than glucose [39]. Therefore, increases in intracellular fructose promote AGE formation [40]. In our research, we attempted to suppress protein saccharification by inhibiting aldose reductase. Administration of the aldose reductase inhibitor Solvinyl to streptozotocin-induced diabetic rats reduced AGEs in skin collagen [41]. Moreover, the pentosidine-like fluorescence (335/385 nm) of the crystalline lens of galactosaemic rats was suppressed by treatment with the aldose reductase inhibitor sorbinin [42]. Administration of an aldose reductase inhibitor to patients with diabetes reduces the amount of N-epsilon-(carboxymethyl)-lysine in erythrocytes [43]. Following the development of many aldose reductase inhibitors, epalrestat was used clinically [44].

Fructose generated from such a polyol pathway is converted to fructose-1-phosphate by fructokinase, and fructose-1-phosphate further produces glyceraldehyde by aldolase. AGEs formed from this glyceraldehyde are highly toxic TAGEs. Increases in intracellular fructose, which trigger glyceraldehyde production, are caused not only by the polyol pathway but also by excessive intake of high-fructose syrup, such as high-fructose corn syrup.

Fructose is a natural ketose that is abundant in fruits and honey. However, in recent years, many soft drinks have been produced using high-fructose corn syrup, which is an isomerised sugar, and a relationship between excessive intake of fructose and metabolic syndrome has been reported [45]. Fructose ingested from soft drinks is taken up into cells by passive transport via GLUT5 in the epithelium of the small intestine. In contrast, glucose and lactose-derived galactose are taken up into cells by active transport via sodium-glucose cotransporter 1. Excessive fructose is transported from small intestinal epithelial cells through the portal vein to the liver and the whole body, thereby increasing glyceraldehyde-derived TAGEs. As discussed later, glyceraldehyde-derived TAGEs generated from fructose can cause liver diseases.
