Lysinoalanine

Figure 13.5 Formation of lysinoalanine nucleophilic additions of the e-amino group of the protein-bound lysine to the double bond of DHA residue (a) causes crosslinking of the polypeptide chain (b) lysinoalanine (c) is formed after hydrolysis.

Friedman, M. and Pearce, K.N. (1989). Copper(II) and cobalt(II) affinities of ll- and LD-lysinoalanine diastereomers Implications for food safety and nutrition, J. Agric. Food Chem., 31, 123-127. 

Pearce, K.N. and Friedman, M. (1988). Binding of copper(II) and other metal ions by lysinoalanine and related compounds and its significance for food safety, J. Agric. Food Chem., 36, 707-717. 

V-1 from acid and alkaline hydrolyzates, SCX-HPLC of amino acids, a mixture of purified crosslinks and hydroxylysine b purified cross-link V-2 c amino acids from an acid hydrolyzate (6 M HCl) of reduced bovine dentin retained on a phenylboronate agarose column after purification as high molecular weight fractions by repeated size exclusion chromatography d as c, alkaline hydrolyzate (2 M KOH). Injections (c, d) resulted from 18 and 52 mg collagen originally hydrolyzed, respectively. 1 = 111 (HP) 2 = V-2 3 = IV 4 = V-1-1 (DHLNL) 5 = HLNL (bovine tendon) 6 = VI (histidinoalanine ) 7 = hydroxylysine 8 = VI (lysinoalanine). 

Relative mobility (Rf - value) for each sample is given FMOC = precolumn derivatization with FMOC-Cl, unident. = unidentified peak, not sign. = not significant peak, - = no fluorescent peak, DHLNL = dihydroxylysinonorleucine, HAL = histidinoalanine, HP = hydroxylysylpyridinoline, LAL = lysinoalanine. 

In addition, peak VI (fig. 1) contained two compounds, one identified as lysinoalanine (table 1). Lysinoalanine is a well-known artefact of alkaline protein treatment but is supposed to be formed in dentin by the reaction between a collagen lysine- and a phosphoprotein phosphoserine residue (Fujimoto et al., 1981). Both compounds were not detected by HPLC after FMOC-derivatization, most likely because of fluorescence quenching inherent to the close vicinity of several FMOC groups attached to one molecule. Thus the unknown compound seems rather similar to lysinoalanine. We suggest the unknown compound is histidinoalanine, which is present in dentin (Fujimoto et al., 1982) and likely shows fluo-rence quenching in its FMOC derivate. 

Fujimoto D, Hirama M and Iwashita T (1981) Occurrence of lysinoalanine in calcified tissue collagen. Biochem Biophys Res Comm 103, 1378-1383. 

De Groot and Slump (40) studied the influence of alkali on soy protein isolates, monitoring the production of lysinoalanine and changes in amino acid content. They found that above pH 10, treatment at 40 C for 4 hours resulted in decreased cystine and increased LAL (Figure la). They also found that at pH 12.2 for 4 hours, lysine and cystine content steadily decreased with increasing temperatures from 20° to 80 C, and LAL content increased dramatically. At pH 12.2 and 4O C they reported that the greatest loss in cystine and increase in LAL occurred in the first hour (Figure lb). Thus they concluded that exposure of soy protein isolate at pH 12.2 for only a short time would destroy some cystine and decrease the nutritive value. 

Postsynthetic modifications of cytoskeletal microfilaments can also occur. For example, epidermal keratin has been found to contain lanthionine, (y-gluta-myllysine) and lysinoalanine, both presumably arising from crosslinkages.306... 

Erbersdobler, et al. (6) found karyocytomegaly in the kidneys of rats fed 16200 to 23300 ppm fructose-lysine in the diet. The diet did not contain lysinoalanine (Fig. 2) but the renal damage was similar to that found after lysinoalanine administration by other authors. The details of this question are reviewed elsewhere (7). Erbersdobler s report has not been confirmed with new data by others. 

Figure 2. Formation of lysinoalanine and other amino acid derivatives from proteins.

Friedman290 reports earlier work by his group, which showed that the presence of glucose during alkaline treatment of soybean proteins significally lowered the amount of lysinoalanine formed. 

Several papers have already linked these discoveries with foods. In the first, CML was found in rat urine, with excretion rates of 4-19%.411 The excretion of CML was definitely related to the intake of CML, rather than to the intake of fructoselysine or lysinoalanine, although adding fructoselysine free of CML did increase the CML excreted, so fructoselysine must be being metabolised to CML or converted into it in the diet before consumption. CML had previously been found in the urine, in quite a high proportion, of premature infants and of hospitalised youngsters, without a clear indication as to its source.413... 

Pentosidine, too, has been found in foods.354 Ion-exchange chromatography with direct fluorescence detection gave a detection limit lower than 50 fig kg-1 protein. The levels in food ranged from not detectable to 2-5 mg kg-1 protein for sterilized and evaporated milk and up to 35 mg kg-1 protein for some bakery products and coffee, a range of concentrations comparable with those in plasma and urine. Pentosidine also increased with storage, but, compared with the crosslinks due to lysinoalanine and histidinoalanine (up to 3000 mg kg-1 protein), it does not play a major part in crosslinking food proteins. 

Other conversions to unnatural residues occur when most proteins are exposed to high pH (80, 81,82). The high pH causes a -elimination of a cystine (see Figure 16) or O-substituted serine or threonine, with the formation of a dehydroalanine or a dehydro-a-aminobutyrate. Such products are subject to nucleophilic attack by the e-amino group of a lysine to form a cross-linkage, such as lysinoalanine, or attack by cysteine to form lanthionine. Walsh et al. (81) have taken advantage of the formation of these cross-links to produce avian ovomucoids that have nonreducible cross-links and have lost the antiprotease activity of one of their two inhibitory sites (see Figure 17). 

Alkaline treatments lysinoalanine (lysine) (100) lanthionine (cysteine) (100) Oxidation Products of Methionine (59-62,70-72) methionine sulfoxide 0 (rats) partly (chicks) 0 (rats) partly (chicks) 90-100 90-100 kidneys liver,... 

Nutritional Effects Due to the Presence of the Maillard Products. Many physiological or antinutritional effects have been attributed to the Maillard products. Specific effects have been attributed to the Amadori products deoxyfructosylphenylalanine (a model substance not likely to be present in large quantities in foods) appears to depress the rate of protein synthesis in chicks (32) and to partially inhibit in vitro and in vivo the absorption of tryptophan in rats (33). The compound e-deoxyfructosyllysine inhibits the intestinal absorption of threonine, proline, and glycine and induces cytomegaly of the tubular cells of the rat kidneys (34) as does lysinoalanine. In parenteral nutrition the infusion of the various Amadori compounds formed during sterilization of the amino acid mixture with glucose is associated with milk dehydration in infants and excessive excretion of zinc and other trace metals in both infants and adults (35,36,37). 

Figure 2. Whole-body autoradiographies of rats, 24 h after oral ingestion of derivatives of U-14C-i.-lysine lysinoalanine, e-deoxyfructosyllysine, e-formyl-lysine, e-(y-glutamyl)lysine, and a-formyllysine.

Whole-Body Autoradiography. Whole-body autoradiography performed 8 h after intravenous injection of e-deoxyfructosyl-U-14C-lysine shows that the radioactivity is localized in the bladder and in the kidneys since the excretion is not complete, and a little in the pancreas (41). Twenty-four h after oral ingestion, the radioactivity is localized mainly in the large intestine and a little in the cortex of the kidneys, giving the same pattern as 14C-lysinoalanine (see Figure 2). 

The most important modifications are the formation of lysinoalanine and lanthionine. Lysinoalanine can be detected or measured by TLC (95) or ion-exchange chromatography (96,97). Lanthionine, which is eluted at the same time as glycine by ion-exchange chromatography (1,2) can be detected by TLC (98). Lysinoalanine was formed also upon heat treatments and has been detected in many heated-food proteins (95). The problems linked to the presence of this amino acid consequently have been generalized to all the heated-food proteins. 

The nutritional and physiological effects of the alkali-treated food proteins have been studied extensively. Efforts have been concentrated mainly on the effects of lysinoalanine. 

Nutritional and Physiological Effects of Alkali-Treated Proteins. The first effect of the alkaline treatment of food proteins is a reduction in the nutritive value of the protein due to the decrease in (a) the availability of the essential amino acids chemically modified (cystine, lysine, isoleucine) and in (b) the digestibility of the protein because of the presence of cross-links (lysinoalanine, lanthionine, and ornithinoalanine) and of unnatural amino acids (ornithine, alloisoleucine, / -aminoalanine, and D-amino acids). The racemization reaction occurring during alkaline treatments has an effect on the nitrogen digestibility and the use of the amino acids involved. 

These effects were described by de Groot and Slump (99) followed by Provansal et al. (94). Later, it was confirmed that the lysine moiety of lysinoalanine was completely unavailable to the rat and only partly available to the chick, while a certain part of the cysteine moiety of lanthionine (32% to 52% according to the racemic mixture) was available to chicks (100) (see Table I). 

The most outstanding effect of the alkali-treated proteins that has been reported is due to the presence of lysinoalanine, which induces renal alterations in rats, designated nephrocytomegaly, which consists of enlarged nuclei and increased amounts of cytoplasm in epithelial cells of the straight portion of the proximal tubules (101,102,103,105,106). This effect has never been found in other species such as mice, hamsters, quails, rabbits, dogs, and monkeys (103). 

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