**4.3 Furosine**

Amadori compounds are measured as furosine (ε-N-2-furoylmethyl-L-lysine) (Figure 5). The content of furosine present in foods is influenced by the kind of heat treatment and/or the storage time. Levels of furosine tend to decline after prolonged storage or after overheating to give rise to other compounds such as CML (Delgado-Andrade *et al*., 2005; Friedman, 1996; Rufiàn-Henares *et al*., 2009).

Fig. 5. Chemical structure of furosine

Furosine is the most specific and important indicator of the initial phase of the Maillard reaction. It is widely used in the analysis of cereal products, since lysine is the limiting amino acid of this product and, thus the presence of furosine is an important marker of protein biological value loss. Monitoring furosine formation and contents helps tailor the processing conditions in order to guarantee the maintenance of the nutritional value of food products (Rufiàn-Henares *et al.*, 2004, 2006; Resmini *et al*., 1990).

Regarding analytical procedures, in 1992, an ion pairing HPLC based methodology was proposed and successfully applied in a series of studies. In 1996, when furosine became commercially available, Reverse Phase-HPLC became the method of choice for furosine analysis. Due to the possible transformation of CML into furosine during heating it is necessary for the acid hydrolysis to be performed in an inert atmosphere, which impairs furosine degradation (Erbersdobler & Somoza, 2007).

#### **4.4 Nε-(carboxymethyl)lysine**

288 Food Industrial Processes – Methods and Equipment

Hydroxymethylfurfural (HMF) is an intermediate compound formed during the Maillard reaction and by the degradation of hexoses at high temperatures at acid conditions (Figure

> O OH O

Spectrophotometric (colorimetric) methods, which are the most usual methods for HMF determination in food, are of limited accuracy since other chromophores in foods may absorb radiation in the same wavelength region, interfering with the results. In addition, colorimetric methods have low sensitivity. Chromatographic methods (liquid or gas high resolution chromatography) are more accurate and sensitive for this purpose, and one of the major advantage of the use of chromatographic methods is the individual determination of HMF and furfural, what can not be achieved by spectrophotometric methods (Erbersdobler & Somoza,

HMF formation is directly linked to the heat intensity applied to food, and because is not usually present in raw and fresh foods, it is considered a thermal damage marker for products containing high carbohydrate concentrations. Moreover, it can be used to monitor the thermal process applied to several different food products such as: breakfast cereals containing dried fruits; caramel and honey; pasta and bakery products (Rufiàn-Henares &

Amadori compounds are measured as furosine (ε-N-2-furoylmethyl-L-lysine) (Figure 5). The content of furosine present in foods is influenced by the kind of heat treatment and/or the storage time. Levels of furosine tend to decline after prolonged storage or after overheating to give rise to other compounds such as CML (Delgado-Andrade *et al*., 2005; Friedman, 1996;

2NH OH

O

**4.2 Hydroxymethyfurfural** 

4) (Arribas-Lorenzo & Morales, 2010).

Fig. 4. Chemical structure of HMF

**4.3 Furosine** 

Rufiàn-Henares *et al*., 2009).

Fig. 5. Chemical structure of furosine

2007; Morales *et al.*, 1997; Rufiàn-Henares *et al.*, 2001).

Delgado-Andrade, 2009; Rufiàn-Henares *et al.,* 2006).

<sup>O</sup> NH

O

Nε-(carboxymethyl)lysine (CML) is a stable, low reactivity advanced Maillard product (Figure 6). CML can be produced by Amadori compound degradation, such as Nε- (fructosyl)lysine (FL). FL undergoes oxidation to form, Nε-(carboxymethyl)lysine (CML). Rdicarbonyls such as glyoxal (GO), formed during the oxidation of the sugar or the Amadori rearrangement products are immediate precursors of CML. Lipid peroxidation is another route to CML, and GO has been suggested as an intermediate. CML is one of the more important markers of bioactive Maillard products and its content is usually correlated to the health risk of ingestion of heat-treated foods (Charissou *et al.*, 2007).

Fig. 6. Chemical structure of CML

The three main metodologies proposed to evaluate CML in foods are: a) RP-HPLC (Reverse Phase/ High Performanc Liquide Chromatography), b) GC/MS (Gas Chromatography connected to Mass Spectrometry) following methylation of the carboxylic group and acylation of the amine group and c) enzyme- linked immunosorbent assay based on a monoclonal anti-CML antibody (Charissou *et al.*, 2007).

Currently, CML analyses in foods are performed by specific immunosorbent (AGE-ELISA-Enzyme Linked Immuno Sorbent Assay). This test is suitable for quantitative CML analysis both in biological samples and food (Goldberg, 2004).

Among the Maillard reaction products, CML is the best characterized end product. It is employed as an advanced glycation end products/advanced lipoxidation end products (AGEs/ALEs) markers in research (Goldberg, 2004).

activation of the receptor for AGEs in the retina could play a significant role in the initiation and progression of age-related macular degeneration and cataracts (Pawlak *et al.*, 2008). Kalousová *et al.* (2002) and most recently, Mostafa *et al.* (2007) showed that AGEs level in plasma proteins are elevated in patients with diabetes. The high blood glucose levels favor the occurrence of spontaneous reactions (glycation) between glucose and proteins, resulting in the formation and excessive deposition of AGEs (Magalhães *et al.*, 2008). In patients with renal failure AGEs accumulation occurs due to the decrease in the extent of degradation and elimination from the body and, also, to increased exposure to oxidative stress. On the other hand, the AGEs and products derived from the process of oxidation promote damage in the renal tissue, leading to greater accumulation of AGEs, creating a vicious cycle (Hartog *et al.*, 2007). The increase in consumption of heated, cooked or roasted food of AGEs

285

Fig. 7. AGE-RAGE interaction and its association with atherosclerosis (Based on Hartog et

Among the mechanisms by which AGEs may contribute to the development and progression of vascular complications of diabetes, is the interaction of these compounds with receptors on the surface of various cell types, such as RAGEs (Receptors for Advanced Glycation End Products) (Marchetti, 2009). The AGE-RAGE interaction in the endothelial cells activates the transcription of nuclear factor-kappaB (NF-κB), with the induction of proinflammatory cytokines, such as the tumor necrosis factor (TNF), interleukin-1 (IL-1), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1) and enhances the expression of the vascular cell adhesion molecule-1 (VCAM-1) (Basta, 2008; Magalhães *et al*., 2008; Méndez *et al*., 2010; Muscat *et al*., 2007). In addition, this interaction in monocytes induces their activation to macrophages and promotes monocyte chemotaxis, and in smooth muscle

accumulation.

al., 2007).
