**2.1 Hyperglycaemia – the basic knowledge on Louis Maillard's discovery**

Glycation (non-enzymatic N-glycosylation) is an endogenous process that contributes to the post-translational modification of proteins. It is slow under normal physiological conditions, giving rise to the presence of lysine- and arginine-derived glycation adducts in cellular and extracellular proteins. Inside cells, the impact of glycation is countered by high turnover and short half-life of numerous cellular proteins. In long-lived extracellular proteins, however, glycation adducts accumulate with age (Sell et al., 1996). Then, some of these adducts may be removed by enzymatic repair mechanisms, whilst all are removed by degradation of the glycated proteins. Degradation of extracellular glycated proteins requires specific recognition by receptors, internalisation and proteolytic processing. There are specific receptors, AGE receptors, which fulfill this role (Thornalley, 1998).

The Maillard reaction is named after Louis Maillard, who discovered over 80 years ago that some amines and reducing carbohydrates react to produce brown pigments (Ellis, 1959). The Maillard reaction proceeds via three major stages (early, advanced and final stage) and is dependent upon factors such as pH, time, temperature, as well as type and concentrations of reactants. Maillard reactions occur both *in vivo* and *in vitro*, and are associated with the chronic complications of diabetes, aging and age-related diseases (Edeas et al., 2010). The first step of this reaction typically involves the nucleophilic addition of a reducing sugar to a primary amine group (e.g. as found on a lysine or at the N-terminus of a protein). In this stage a reversible Schiff base is formed, which can undergo a slow irreversible rearrangement to form more stable Amadori product that accumulates over time (Fig. 1). The total amount of such accumulated products is known to be dependent on the type of sugar that is causing the glycation, the incubation time and sugar concentration, as well as the type protein that is being modified (Barnaby et al., 2011).

Fig. 1. General scheme of Maillard pathways in diabetic organism.

The first product of Maillard reaction is a simple glycosylamine, which readily undergoes the Amadori rearrangement to produce 1-amino-1-deoxy-2-ketoses. The large body of literature on these reactions is due to the multitude of possible reaction pathways and products, including fragmentations of the carbohydrates and formation of aromatic compounds from cyclisation/dehydration processes (Hodge, 1955). Reducing disaccharides also undergo this reaction, and it is a well-documented process for the degradation of lactose during the heating of milk. Reducing carbohydrates such as glucose, maltose, and lactose are tautomers and are in equilibrium with their more reactive aldehyde forms; nonreducing carbohydrates, such as mannitol, sucrose and trehalose, are not subject to Maillard reactions. Although early scientists believed that only primary aromatic amines were capable to become glycated, subsequent research has shown that nearly all primary and secondary amines, both aromatic and aliphatic, are capable of this reaction (Wirth et al., 1998).

Fragmentation of glucose adducts in early glycation processes establishes many parallel glycation pathways that lead to the subsequent formation of the so-called **A**dvanced **G**lycation **E**nd-products (AGEs). Analogous oxidation and dehydration reactions have been found in glycation by other hexose and pentose derivatives. Glyoxal, methylglyoxal and 3 deoxyglucosone (3-DG)-derived AGEs may be present in proteins glycated by glucose. Methylglyoxal-derived AGEs are common to proteins modified by glucose and by the authentic α-oxoaldehyde. Indeed, similar binding to AGE receptors has been found for these proteins. The formation of α-oxoaldehydes from monosaccharides, Schiff's bases and fructosamines suggests that AGEs may be formed at all stages of glycation (Westwood et al., 1999). AGEs alter structure and functions of proteins. It has been shown that the formation of AGEs *in vivo* contributes to several pathophysiological impairments associated with aging and diabetes mellitus, such as chronic renal insufficiency, Alzheimer's disease, nephropathy, neuropathy and cataract (Ravelojaona et al., 2007).

### **2.2 Impact of AGEs on human organism – undesirable effects on our health**

Glycation of amino-groups on small or large cell constituents induces a number of undesirable effects in a plethora of age-related pathologies, overall referred to as glycation-induced health

The first product of Maillard reaction is a simple glycosylamine, which readily undergoes the Amadori rearrangement to produce 1-amino-1-deoxy-2-ketoses. The large body of literature on these reactions is due to the multitude of possible reaction pathways and products, including fragmentations of the carbohydrates and formation of aromatic compounds from cyclisation/dehydration processes (Hodge, 1955). Reducing disaccharides also undergo this reaction, and it is a well-documented process for the degradation of lactose during the heating of milk. Reducing carbohydrates such as glucose, maltose, and lactose are tautomers and are in equilibrium with their more reactive aldehyde forms; nonreducing carbohydrates, such as mannitol, sucrose and trehalose, are not subject to Maillard reactions. Although early scientists believed that only primary aromatic amines were capable to become glycated, subsequent research has shown that nearly all primary and secondary amines, both aromatic and aliphatic,

Fragmentation of glucose adducts in early glycation processes establishes many parallel glycation pathways that lead to the subsequent formation of the so-called **A**dvanced **G**lycation **E**nd-products (AGEs). Analogous oxidation and dehydration reactions have been found in glycation by other hexose and pentose derivatives. Glyoxal, methylglyoxal and 3 deoxyglucosone (3-DG)-derived AGEs may be present in proteins glycated by glucose. Methylglyoxal-derived AGEs are common to proteins modified by glucose and by the authentic α-oxoaldehyde. Indeed, similar binding to AGE receptors has been found for these proteins. The formation of α-oxoaldehydes from monosaccharides, Schiff's bases and fructosamines suggests that AGEs may be formed at all stages of glycation (Westwood et al., 1999). AGEs alter structure and functions of proteins. It has been shown that the formation of AGEs *in vivo* contributes to several pathophysiological impairments associated with aging and diabetes mellitus, such as chronic renal insufficiency, Alzheimer's disease, nephropathy,

**2.2 Impact of AGEs on human organism – undesirable effects on our health** 

Glycation of amino-groups on small or large cell constituents induces a number of undesirable effects in a plethora of age-related pathologies, overall referred to as glycation-induced health

Fig. 1. General scheme of Maillard pathways in diabetic organism.

are capable of this reaction (Wirth et al., 1998).

neuropathy and cataract (Ravelojaona et al., 2007).

hazards and including cardiovascular diseases, kidney insufficiencies, retinopathy, or effects of AGEs on embryonic development, as observed in diabetes-related gravidities. As such reactions proceed with a speed proportional to the concentrations of the interacting substances, hyperglycaemia is an important factor for its acceleration (Urios et al., 2007).

It is very important to remember that Maillard products derive also from ''ready made'' ingested food. Table 1 shows the contents of Maillard products in some foods (expressed as Nε-carboxylysine), as selected from data published by Goldberg et al. (2004).


\*AGE denotes N-carboxymethyllysine (CML)-like immunoreactivity, assessed by enzyme-linked immunosorbent assay using monoclonal antibody 4G9.

Table 1. The content of Maillard products in the selected victuals.

The Maillard reaction between reducing sugars and amino acids is a common reaction in foods, which undergo thermal processing. Desired consequences, like the formation of flavor and brown color of some cooked foods, but also the destruction of essential amino acids and the production of anti-nutritive compounds, require to consider the relevant mechanisms for controlling of Maillard reaction intermediates and final products. Processes such as roasting, baking or frying rely on favorable effects of the Maillard reaction, such as color and flavor formation, whereas during drying, pasteurisation and sterilisation the occurrence of the Maillard reaction is unfavorable. Nutritional losses of essential amino acids that are involved in the reaction, as well as the formation of reaction products are among those unwanted effects (Jaeger et al., 2010).

There is a limited number of studies that have been used to investigate the health effects of dietary Maillard neoformed compounds in humans. Some observational studies have been carried out to address the question of absorption, biodistribution and elimination of dietary Maillard Reaction Products (MRP), and to observe the associations between food exposure to MRPs and their *in vivo* levels.

Some reports have shown that in tobacco leaves, which are dried in the presence of sugars, the Maillard reaction cascade leads to a formation of glycated and oxidative derivatives. These compounds are inhaled during the smoking, after that they are absorbed by lungs and conjugated with serum proteins. It was evidenced that total serum AGE level in cigarette smokers is significantly higher in comparison with non-smokers. However, the highest level of AGEs was detected in the arteries and ocular lenses in diabetic smokers (Vlassara & Palace, 2002).

Furthermore, high AGE levels were observed in industrially preprocessed foods from animal products, like frankfurters, bacon, and powdered egg whites, compared with the unprocessed forms. Across all categories, exposure to higher temperature most of all raised the AGE content (for equal food weights). The temperature level appeared to be more critical than the duration. Also, microwaving increased AGE content more rapidly compared with conventional cooking methods (Peppa et al., 2002). Based on the above data, it is well evidenced that dietary glycoxidation products may constitute an important link between the increased consumption of animal fat and meat and the subsequent development of diabetic complications. However, the problem of AGEs' presence in food is well known, and therefore presently scientists call to use diets containing low contents of these compounds undesirable for out health.

Paradoxically, because of the metabolic demands of the brain, the human body has an obligatory requirement for glucose, approaching 200 g/day. The blood glucose concentration is tightly regulated by homeostatic regulatory systems and maintained between 40 mg/dl (2.2 mmol/l) and 180 mg/dl (10.0 mmol/l). Hypoglycaemia below the lower limit may result in coma, seizures, or even death. Hyperglycaemia, exceeding the upper limit, is associated with immediate glycosuria and caloric loss, as well as long-term consequences, like retinopathy, atherosclerosis, renal failure, etc. Under normal physiological conditions hyperglycaemia stimulates insulin secretion, promoting uptake of glucose by muscles and adipose tissue (Chiu & Taylor, 2011).

Nevertheless, several studies suggest that some MRPs present in foods could have beneficial effects on human health. For instance, the melanoidins are brown Maillard polymers, which seem to have functional properties in food products and are also capable of inhibiting growth of a tumour cell line in culture (Marko et al., 2003). In addition, it was also found recently that a selection of foods rich in MRPs could inhibit the oxidation of LDL *in vitro*.

The Maillard reaction between reducing sugars and amino acids is a common reaction in foods, which undergo thermal processing. Desired consequences, like the formation of flavor and brown color of some cooked foods, but also the destruction of essential amino acids and the production of anti-nutritive compounds, require to consider the relevant mechanisms for controlling of Maillard reaction intermediates and final products. Processes such as roasting, baking or frying rely on favorable effects of the Maillard reaction, such as color and flavor formation, whereas during drying, pasteurisation and sterilisation the occurrence of the Maillard reaction is unfavorable. Nutritional losses of essential amino acids that are involved in the reaction, as well as the formation of reaction products are

There is a limited number of studies that have been used to investigate the health effects of dietary Maillard neoformed compounds in humans. Some observational studies have been carried out to address the question of absorption, biodistribution and elimination of dietary Maillard Reaction Products (MRP), and to observe the associations between food exposure

Some reports have shown that in tobacco leaves, which are dried in the presence of sugars, the Maillard reaction cascade leads to a formation of glycated and oxidative derivatives. These compounds are inhaled during the smoking, after that they are absorbed by lungs and conjugated with serum proteins. It was evidenced that total serum AGE level in cigarette smokers is significantly higher in comparison with non-smokers. However, the highest level of AGEs was detected in the arteries and ocular lenses in diabetic smokers (Vlassara &

Furthermore, high AGE levels were observed in industrially preprocessed foods from animal products, like frankfurters, bacon, and powdered egg whites, compared with the unprocessed forms. Across all categories, exposure to higher temperature most of all raised the AGE content (for equal food weights). The temperature level appeared to be more critical than the duration. Also, microwaving increased AGE content more rapidly compared with conventional cooking methods (Peppa et al., 2002). Based on the above data, it is well evidenced that dietary glycoxidation products may constitute an important link between the increased consumption of animal fat and meat and the subsequent development of diabetic complications. However, the problem of AGEs' presence in food is well known, and therefore presently scientists call to use diets containing low contents of

Paradoxically, because of the metabolic demands of the brain, the human body has an obligatory requirement for glucose, approaching 200 g/day. The blood glucose concentration is tightly regulated by homeostatic regulatory systems and maintained between 40 mg/dl (2.2 mmol/l) and 180 mg/dl (10.0 mmol/l). Hypoglycaemia below the lower limit may result in coma, seizures, or even death. Hyperglycaemia, exceeding the upper limit, is associated with immediate glycosuria and caloric loss, as well as long-term consequences, like retinopathy, atherosclerosis, renal failure, etc. Under normal physiological conditions hyperglycaemia stimulates insulin secretion, promoting uptake of

Nevertheless, several studies suggest that some MRPs present in foods could have beneficial effects on human health. For instance, the melanoidins are brown Maillard polymers, which seem to have functional properties in food products and are also capable of inhibiting growth of a tumour cell line in culture (Marko et al., 2003). In addition, it was also found recently that a selection of foods rich in MRPs could inhibit the oxidation of LDL *in vitro*.

among those unwanted effects (Jaeger et al., 2010).

these compounds undesirable for out health.

glucose by muscles and adipose tissue (Chiu & Taylor, 2011).

to MRPs and their *in vivo* levels.

Palace, 2002).

The high diversity of the MRPs formed in the very diverse food matrices makes it impossible to classify all of them as glycotoxins. It is admitted that they have different beneficial or detrimental biological activities. Thus, more well-controlled clinical experiments are needed to establish the role of the ingested MRPs, pure or added to food matrices, following acute or chronic exposures (Tessier & Birlouez-Aragon, 2010).
