Proteins lysinoalanine

Woodard, J. C., Short, D. D., Alvarez, M. R. and Reyniers, J. (1975). Biologic effects of N-e-(DL-2-amino-2-carbOKyethyl)-L-lysine, lysinoalanine. "Protein Nutritional Quality of Foods and Feeds" M. Friedman, Ed., Marcel Dekker, New York, pp. 595-616. 

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.

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. 

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. 

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. 

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). 

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). 

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. 

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). 

After some controversies concerning the effects of the diet composition and the strain of the animals on the development of nephrocyto-megaly, it was admitted that free as well as protein-bound lysinoalanine were really responsible for this effect. It also was shown that this nephro-cytomegaly was reversible (109) and that the dose response was not the same for the different stereoisomers of lysinoalanine (107,108,110) the ld isomer is more active than the ll, dl, and dd isomers. 

Metabolic Transit of Lysinoalanine. Urinary and Fecal Excretion of Protein-Bound Lysinoalanine (113). Three different alkali-treated proteins (lactalbumin, fish protein isolate, and soya protein isolate) containing, respectively, 1.79, 0.38, and 0.14 g of lysinoalanine/16 g nitrogen were given to rats and the urines and feces were collected. Lysinoalanine was measured before and after acid hydrolysis. The fecal excretion varied from 33 to 51% of the total ingested lysinoalanine and the urinary excretion varied from 10 to 25%. The higher level of lysinoalanine found after acid hydrolysis indicates that a certain quantity is excreted in the urines as combined lysinoalanine (see Table VII). The total recovery was inferior to the ingested quantity (50 to 71%) indicating that the molecule is transformed or retained in the body of the rat. 

Table VII. Urinary and Fecal Excretions of Protein-Bound Lysinoalanine in Rats° (percentage of ingested quantity) (113)...

Alkali has long been used on proteins for such processes as the retting of wool and curing of collagen, but more recently it has received interest from the food industry. Alkali can cause many changes such as the hydrolysis of susceptible amide and peptide bonds, racemization of amino acids, splitting of disulfide bonds, beta elimination, and formation of cross-linked products such as lysinoalanine and lanthionine. 

Nashef et al. (41) also reported that the rate of 6 elimination from cystine was directly dependent on hydroxide ion concentration although the relationship was not linear perhaps because of the complexity of the reaction (Equation 7). Sternberg and Kim (20.) found the rate of lysinoalanine formation in casein to be dependent on hydroxide ion concentration. Touloupais and Vassiliadis (45) also found the rate of lysinoalanine formation in wool to be pH dependent. These workers did not measure the rate of 6 elimination, therefore the rate determining step is not known. These results on proteins appear to be in contradiction to those of Samuel and Silver (46) who reported that hydroxide ion concentration had no effect on the rate of 6 elimination from free phosphoserine between pH 7 and 13.5. Because of the effect... 

The reactions of proteins in alkaline solution are very important from a number of standpoints. We have already discussed several uses of alkali treatment in food processing in the introduction. When contact between the food and alkali is kept to a minimum at the lowest temperature possible with adequate control of mixing, etc. there is presently no apparent reason to discontinue its use. Low levels of lysinoalanine occur in food which has been processed in the absence of added alkali, even at pH 6 and in the dry state (20). For example, the egg white of an egg boiled three minutes contained 140 ppm of lysinoalanine while dried egg white powder contained from 160 to 1820 ppm of lysinoalanine depending on the manufacturer (20). No lysinoalanine was found in fresh egg white, 3 Elimination and addition of lysine to the double bond of dehydroalanine reduce the level of the essential amino acid lysine. This can be prevented by adding other nucleophiles such as cysteine to the reaction. Whether lysinoalanine (and other compounds formed by addition reactions) is toxic at low levels in humans is not known. 

Figure 1. Postulated mechanism of racemization and lysinoalanine formation via a common carbanion intermediate. Note that two B-elimination pathways are possible (a) a concerted, one-step process (A) forming the dehydroprotein directly and (b) a two-step process (B) via a carbanion intermediate. The carbanion, which has lost the original asymmetry, can recombine with a proton to regenerate the original amino acid residue which is now racemic. Proton transfer may take place from the environment of the carbanion or from adjacent NH groups, as illustrated. Protein anions and carbanions can also participate in nucleophilic addition and displacement reactions (24, 82, 83).

Since lysinoalanine and at least one D-amino acid are toxic to some animals (35), we wished to distinguish their effects in alkali-treated proteins. Such discrimination is possible, in principle, since we have found that acylating the e-amino group of lysine proteins seems to prevent lysinoalanine formation. Since lysinoalanine formation from lysine requires participation of the e-amino group of lysine side chains, acylation of the amino group with acetic anhydride is expected to prevent lysinoalanine formation under alkaline conditions if the protective effect survives the treatment. This is indeed the case (16). 

Although lysinoalanine concentrations in foods are usually lower than amounts needed to induce nephrocytomegaly in rats, health hazard may exist since human tolerances for the "unnatural" amino acids generated during commercial processing are not known. If the protein-induced lesion is not precancerous, it is still important to understand its etiology. 

Friedman, M. (1978). Inhibition of lysinoalanine synthesis by protein acylation. In "Nutritional improvement of Pood and Peed Proteins , M. Friedman, Ed., Plenum Press, New York, pp. 613-648. 

De Groot, A. P., Slurp, P., Feron, V. J. and Van Beek, L. (1976). Effects of alkali-treated proteins feeding studies with free and protein-bound lysinoalanine in rats and other animals. J. Nutr. 106, 1527-1538. 

Gould, D. H. and MacGregor, J. T. (1977). Biological effects of alkali-treated protein and lysinoalanine an overview. In "Protein Crosslinking Nutritional and Medical Consequences," M. Friedman, Ed., Part B,... 

Struthers, B. J., Dahlgren, R. R. and Hopkins, D. T. 1976. Biological effects of feeding graded levels of alkali hydrolyzed soy protein containing lysinoalanine... 

Karayiannis, N. (1976). Lysinoalanine Formation in Alkali Treated Proteins and their Biological Effects, Ph.D. Thesis. University of California, Berkeley. 

O Donovan, C. J. (1976). Recent studies of lysinoalanine in alkali-treated proteins. Fd. Cosmet. Toxicol 14, 483-489. 

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