Furosine method

Detection of Amadori Products Various methods are now available for the detection and quantification of Amadori products in body proteins (Figure 3). The furosine method, developed by Finot et al. (15), allows the quantification of glycosylated lysine residues. It has been used recently by Dolhover, et al. 

This leads to a decrease in the biological value of the protein. Milk is a good model to study the nutritional effects of the Maillard reaction because of its high levels of lysine and lactose. In processed milks, unavailable lysine is present only as e-deoxylactulosyllysine (9,24) its level which depends on the type of treatment can be measured by the furosine method (24,31). 

In the advanced and late "Maillard reaction" the furosine method is not valuable since the Amadori products lead to further compounds. Therefore the amount of available lysine in severely damaged foods is underestimated. In the case of fructose rich food (e.g. special diets for diabetics or products which are sweetened with honey or concentrated fruit juice) similar false conclusions are possible since glucoselysine which is formed from fructose during the "Maillard reaction" is not a precursor for furosine. 

Several methods have been developed for assaying non-enzymatic glycosylation. As far as biological systems are concerned, these have been extensively reviewed by A. J. Furth in 1988 (5). They include both assays on intact proteins after chemical degradation and selective detection of e.g. 5-hydroxymethylfurfural (HMF) and formaldehyde using the thiobarbituric assay (TEA), and assays on protein hydrolysates with or without previous reduction of the protein-bound Amadori compound. In this last case, the analysis is based on the determination of furosine which is specifically formed from lysine Amadori compounds with a yield of approximately 30% (6). The furosine method, originally developed for milk (7), has been the subject of several analytical improvements both for food products (8) and biological materials (9). More recently, another method has been proposed to evaluate the extent of early Maillard reaction in milk products. This method is based on direct measurement of the Amadori product lactuloselysine which is released after complete enzymatic hydrolysis (10). 

Fig. 7. Correlation between ALV n and available lysine calculated from % blocked lysine via the furosine method.

The furosine method (FUR) (Fig. 7). In milk samples, this method allows the direct determination of blocked lysine (DL-Lys) as grammes Lys/16 g N or, still more conveniently, as a percentage of the given theoretical lysine value when the nitrogen content of the sample... 

Obviously, available lysine can then be calculated from blocked lysine, taking the theoretical lysine value as 100. In this case, (Fig. 7), the correlation equation with ALV n has no special meaning, since this regression was used to validate the furosine method. In our experience, the furosine method has been very useful for routine control of lysine blockage in industrial milk samples. It should be borne in mind, however, that the method as described can only be used to determine early Maillard damage to lysine (deoxyketosyl form). 

Vallejo-Cordoba, B., Mazorra-Manzano, M. A., and Gonzalez-Cordova, A. R, New capillary electrophoresis method for the determination of furosine in dairy products, J. Agric. Food Chem., 52, 5787, 2004. 

In heat treated or stored food products several amino acids are not fully available because of derivatization or crosslinking reactions. Since 30 years furosine is known as a useful indicator of early Maillard reaction which is applied in food science, nutrition and medical biochemistry. Recently more sensitive analytical methods for furosine determination are available which have again increased the attractivity of this important indicator. Lately, N -carboxymethyllysine (CML) became available as another marker of special interest, because CML is a more useful indicator of the advanced heat damage by Maillard reaction than furosine. In addition, CML has the advantage to indicate reactions of lysine with ascorbic acid or ketoses such as fructose. Indicators for protein oxidation of sulfur amino acids are methionine sulfoxide and cysteic acid. An established marker for cross-linking reactions is lysinoalanine, which also indicates protein damages due to processing under alkaline conditions. Other markers formed as a consequence of alkaline treatment are D-amino acids. 

In dairy products also hydroxymethylfurfural (HMF) and lactulose are common markers. For the determination of HMF, which also results from the Maillard condensation (18), precursors of browning products in milk are transformed to HMF after addition of oxalic acid and following heating (79). Principally, the HMF value of a milk can be used as an indicator for the heating process, but data from literature offer a wide range for this value which suspect that the HMF determination is insufficiently reproducible between laboratories. In particular, the level of HMF in untreated material, measured during the determination of "total HMF" and subtracted from the levels in treated milks, is a source of variation (20). However a comparison between the furosine and the HMF-method demonstrated the usefulness of the HMF-method as a rapid and simple measure of heat damage caused by the UHT process (77). 

Some years ago consumers were disturbed by a report that noxious compounds including D-amino acids are produced in microwaved food (56). However, several authors found afterwards that there are no differences in the content of D-amino acids between the microwave heating and conventional cooking methods (57, 38, 59). Also in the concentrations of the above mentioned markers (furosine ect.) no microwave-specific changes in food were found 40,41),... 

The determination oj furosine (LySfu )- This method is based on the fact that lysine blocked in an early Maillard reaction in the form of e-N-deoxyketosyl-lysine yields upon acid hydrolysis a constant proportion of furosine (Fig. 2) (e-N-2-furoylmethyl-L-lysine). The blocked lysine corresponds, then, to 3-1 times furosine. The available lysine value can then be calculated by subtracting the blocked lysine value from the theoretical lysine value (ThL = lysine present in the unheated milk sample). 

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