Metabolic transit of lysinoalanine (LAL) bound to protein and of free radioactive [14C]-lysinoalanine.
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Alanine↗
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Lysinoalanine in food and in antimicrobial proteins.
Heat and alkali treatment of food proteins widely used in food processing results in the formation of crosslinked amino acids such as lysinoalanine, ornithinoalanine, lanthionine, and methyl-lanthionine and concurrent racemization of L-amino acid isomers to D-analogues. The mechanism of lysinoalanine formation is a two-step process: first, hydroxide ion-catalyzed elimination of cysteine and serine residues to a dehydroalanine intermediate; second, reaction of the double bond of dehydroalanine with the epsilon-NH2 group of lysine to form a lysinoalanine crosslink. The corresponding elimination-addition reaction of threonine produces methyl-dehydroalanine, which then reacts with the NH2 and SH groups to form methyl-lysinoalanine and methyl-lanthionine, respectively. The crosslinked amino acids lanthionine and methyl-lanthionine are formed by analogous nucleophilic addition reactions of the SH group of cysteine to dehydroalanine and methyl-dehydroalanine, respectively. Processing conditions that favor these transformations include high pH, temperature and exposure time. Factors which minimize lysinoalanine formation include the presence of SH-containing amino acids such as cysteine, N-acetyl-cysteine, and glutathione, dephosphorylation of O-phosphoryl esters, and acylation of epsilon-NH2 groups of lysine side chains. The presence of lysinoalanine residues along a protein chain decreases digestibility and nutritional quality in rodents but enhances nutritional quality in ruminants. Protein-bound and free lysinoalanines are reported to induce enlargement of nuclei of rat kidney cells. All of the mentioned dehydro and crosslinked amino acids also occur naturally in certain peptide and protein antibiotics. These include duramycin, cinnamycin, epidermin, subtilin and the widely used food preservative nisin. Mechanistic rationalizations are offered for the observed antimicrobial activities of these compounds in relation to their structures. The cited findings and new research to better define the chemistry and dietary and antimicrobial roles of lysinoalanine and related compounds should lead to better and safer foods.
Animals↗
Biological effects of alkali-treated protein and lysinoalanine: an overview.
The renal alterations induced by alkali-treated protein and lysinoalanine are reviewed and their biological implications discussed. Alkali-treated proteins and lysinoalanine, an unusual amino acid formed in proteins during alkali treatment, have been shown to produce a renal alteration characterized by nuclear and cytoplasmic enlargement, with alterations in DNA synthesis, mitotsis and nucleoprotein. These changes are localized in the straight portion of the proximal renal tubule and have been observed in rats but not in several other species. The nephrotoxic effect of synthetic lysinoalanine has been consistently demonstrated, but the ability of alkali-treated protein to induce renal alterations is apparently modified by factors other than lysinoalanine content. Factors which may influence the development of the kidney lesions in animals fed alkali-treatment protein are discussed, including nutritional factors, the chemical form of lysinoalanine in the protein, species differences, and metabolic fate. Other clinical and experimental conditions that result in similar renal alterations are presented for comparison with the lysinoalanine induced lesion, and possible functional consequences are considred.
Alanine↗
Inhibition of lysinoalanine synthesis by protein acylation.
Treating wheat gluten, soy protein, and lactalbumin under alkaline conditions at 65 degree C for various times destroys part of the threonine, cystine, lysine, tyrosine, and arginine residues in these proteins. The losses were accompanied by the appearance of lysinoalanine and unidentified ninhydrin-positive substances. Treating wool under somewhat milder alkaline conditions destroys part of the cystine and lysine residues, but not the other amino acids cited. In this case, loss of cystine and lysine was accompanied by the appearance of lanthionine and lysinoalanine. Amino acid analysis of alkali-treated acylated proteins revealed that acylation by acetic and succinic anhydrides prevents or minimizes destruction of lysine residues and the formation of lysinoalanine in wheat gluten and soy protein and, in wool, minimizes destruction of cystine and lysine residues and the formation of lanthionine lysinoalanine. Mechanisms are proposed to explain the observed inhibiting effects of protein acylation and oertain additives on lysinoalanine formation.
Acylation↗
Studies on the utilization of lysinoalanine and lanthionine.
Studies were conducted to determine the nutritional value of lysinoalanine for rats and chicks and the nutritional value of lanthionine for chicks. For each experiment, purified crystalline amino acid diets and crystalline lysinoalanine and lanthionine were employed. Results indicated that the lysine moiety of lysinoalanine is completely unavailable to the rat and only partially available to the chick. Of 10 rats fed lysinoalanine, seven developed nephrocytomegaly. The lanthionine studies indicated that the cysteine moiety of lanthionine is 32% available when fed as the racemic mixture and 52% available when fed as the L-DL-isomeric mixture. Thus, it appears that lysinoalanine formation in alkali-treated proteins results in a corresponding decrease in nutritionally available lysine. However, the lanthionine formed under similar conditions results in loss of only one-half of the corresponding level of nutritionally available cysteine.
Alanine↗
Inactivation of metalloenzymes by lysinoalanine, phenylethylaminoalanine, alkali-treated food proteins, and sulfur amino acids.
Synthetic lysinoalanine (LAL) may be a more effective inhibitor of the zinc-containing enzyme carboxypeptidase A than is ethylenediamine tetraacetic acid (EDTA). The enzyme is also inactivated by alkali-treated, lysinoalanine-containing food proteins such as casein, high-lysine corn protein, lactalbumin, soy protein isolate, and wheat gluten, and by alkali-treated zein, which contains no lysinoalanine. Zinc sulfate regenerates only part of the enzymatic activity after exposure to the treated proteins. The extent of inhibition increases with protein concentration and time of treatment. Any inhibition due to phytate is distinct from that due to the treatment. Phenylethylaminoalanine (PEAA), derived from biogenic phenylethylamine, inhibited enzymatic activity of the metalloenzyme carboxypeptidase A (CPA). The inhibition was maximal at pH 7.0 in the pH range 7 to 8.5. The extent of inhibition increased with time of treatment and PEAA concentration. N-acetyl-PEAA did not inhibit the enzyme, suggesting that the free alpha-NH2 group is required for inhibition. PEAA, LAL, sodium phytate, and cysteine also inactivated the copper enzyme, polyphenol, oxidase (tyrosinase) which plays a major role in enzymatic (oxidative) browning of foods. Analogous comparative studies with LAL, EDTA, and sodium phytate suggest that the potency of PEAA as an inhibitor of CPA is similar to that of sodium phytate, and that of the four compounds tested, PEAA is least effective against tyrosinase. Related studies of the iron and copper containing enzyme cytochrome C oxidase showed that EDTA was not inhibitory, PEAA was slightly inhibitory, and LAL and sodium phytate were stronger inhibitors. Mechanistic explanations are offered to account for some of these observations. The possible relevance of these findings to in vivo protein digestion, enzymatic (oxidative) browning of foods, and the mechanism of the lysinoalanine effect on kidney cells are also discussed.
Alkaline Phosphatase↗
Lysinoalanine as a metal chelator. An implication for toxicity.
Synthetic lysinoalanine (N epsilon-DL-(2-amino-2-carboxyethyl)-L-lysine) was found to have a strong chelating ability for metals. It became colored when mixed with Cu+2 and showed absorption characteristics typical of a complex. Lysinoalanine could inactivate metalloenzymes such as carboxypeptidases A and B and yeast alcohol dehydrogenase, by removing the zinc ion from the active site. Model building for a mononuclear complex of the metal and lysinoalanine with space-filling models was possible for the LD-isomer, N epsilon-D-(2-amino-2-carboxymethyl)-L-lysine. Etiological studies of its toxicity to humans should be made because the chelating ability of lysinoalanine is sufficiently strong to remove the metal from the enzyme active center at millimolar concentration.
Alcohol Dehydrogenase↗
Analysis of hog thyroglobulin. Identification of galactosamine and absence of lysinoalanine, a potential product of tyrosine coupling.
A small peak in the amino acid analysis of hog thyroglobulin was observed in the region reported for lysinoalanine and galactosamine. Since galactosamine had been previously reported absent in hog thyroglobulin, the possibility that this peak was lysinoalanine, a potential product of the coupling of two iodotyrosines, was investigated. Tests, however, showed that the material was galactosamine and that hog thyroglobulin contains no significant amount of lysinoalanine. Approximately 5 moles of galactosamine were found per mole of thyroglobulin.
Animals↗
Interaction of lysinoalanine with the protein synthesizing apparatus.
Lysinoalanine [N epsilon-(DL-2-amino-2-carboxyethyl)-L-lysine; LAL], a nephrotoxic lysine analog, inhibits the lysyl-tRNA-synthetase (EC 6.1.1.6) of prokaryotic and eukaryotic cells competitively at micromolar concentrations. Incorporation of [14C]lysine into protein by a cell-free eukaryotic protein-synthesizing system was inhibited by LAL. Inhibition was 69.7% and 18.4% at LAL concentrations of 1.0 mM and 0.1 mM, respectively. LAL was incorporated into protein as well as being an inhibitor as indicated by the incorporation of [14C]LAL into protein by the cell-free eukaryote protein-synthesizing system. The proteins labeled with [14C]LAL co-electrophoresed with those labeled with [14C]lysine. These results indicate that LAL is an inhibitor of both prokaryote and eukaryote lysyl-tRNA-synthetase. Furthermore, it is incorporated into protein. Both of these actions can be factors in the nephrotoxicity of this common food contaminant. Possible mechanisms for the toxicity of lysinoalanine are discussed.
Amino Acyl-tRNA Synthetases↗
Lysinoalanine: absence of mutagenic response in the Salmonella/mammalian-microsome mutagenicity assay.
Lysinoalanine (N epsilon-(DL-2-amino-2-carboxyethyl)-L-lysine) was tested for mutagenicity in the Ames Salmonella/mammalian-microsome mutagenicity assay. No mutagenic response was detected at doses up to 5 mg/plate when samples were pre-incubated without S-9 mix, nor when they were pre-incubated with S-9 mix prepared from Aroclor 1254-induced rat liver or kidney. The results indicate that the stereoisomers of lysinoalanine likely to be present in the greatest proportions in processed foods are not mutagenic in the Ames assay.
Animals↗
Lysinoalanine content of formulas for enteral nutrition.
Casein and caseinates are the main ingredients of formulas for enteral nutrition. Their manufacturing procedure and the thermal treatments necessary to assure microbiological stabilization and satisfactory shelf-life of the end-products are particularly favorable for the formation of lysinoalanine (LAL), a cross-linked amino acid that is considered a useful marker of the thermal damage and reduced digestibility of proteins. The lysinoalanine content of 18 different kinds of formulas for enteral nutrition was determined by HPLC after derivatization. The liquid formulas have an average value of 528 microg/g protein LAL, ranging from 160 to 800 microg/g protein (average content of formulas for pediatric use 747 microg/g protein). These values are rather high considering that the average value detected in UHT-treated drinkable milk is 117 microg/g protein. In principle, the preparation of caseinates and the thermal stabilization of the end products are the two steps more favorable for the formation of LAL. The fact that the five samples stabilized by an UHT-treatment have an average value of 512 microg/g protein suggests that the LAL content depends more on the quality of the starting ingredients than on the sterilization process. A better selection of the starting ingredients should improve the quality of formulas for enteral nutrition, which is very desirable when formulating foods for consumers with very high nutritional demands.
Caseins↗
Lysinoalanine formation in wool after treatments with some phosphate salts.
Treatment of wool with solutions of sodium tripolyphosphate, potassium tetrapyrophosphate, and sodium phosphate results in the formation of lysinoalanine, the amount of which increases from the first to the last reagent. As this may be due to the different pH, solutions of the three salts of the same pH were also tried. This series of experiments suggests that although the hydroxyl ions contribute to the formation of lysinoalanine, the governing factor is the kind of the anions concerned, as the sodium phosphate acts in a more rigorous may than the two other salts, in spite of the smaller concentration and the faster reduction of the pH of the solution during treatment.
Alanine↗
[Formation of dehydroalanine, lanthionine and lysinoalanine during heat treatment of beta-lactoglobuline A (author's transl)].
Solutions of beta-lactoglobulin A were heated at 97.5 degrees C at pH 6.9 and 9.8 80 min. The formation of dehydroalanine, lanthionine and lysinoalanine as well as changes in the levels of cysteine, cystine and the basic amino acids were recorded. Lanthionine (equal amounts of the 1- and the mesoform) was formed only in small amounts, while the lysinoalanine levels were rather high. Beside this, lysine was converted to unknown compounds. The main sources for the formation of dehydroalanine are the cystine moieties of the protein. Cysteine residues are first oxidized to cystine before beta-elimination occurs.
Alanine↗
Lysinoalanine: presence in foods and food ingredients.
Lysinoalanine, N6-(DL-2-amino-2-carboxyethyl)-L-lysine, an unusual amino acid implicated as a renal toxic factor in rats, has been found in proteins of home-cooked and commercial foods and ingredients. Although it has been reported to occur in both edible and nonfood proteins only after alkali treatment, it has now been identified in food proteins that had not been subjected to alkali. Lysinoalanine is generated in a variety of proteins when heated under nonalkaline conditions.
Dietary Proteins↗
Selective fortification of lysinoalanine, fructoselysine and N epsilon-carboxymethyllysine in casein model systems.
In the present study, a promising strategy to study nutritional effects of selected chemical reaction products formed in heat treated protein containing foods is addressed. In due course, a selective fortification of different marker compounds for lysine damage in casein-sugar mixtures was performed to provide model systems being applicable to investigate biological effects of the cross-link lysinoalanine (LAL), the MRPs fructoselysine (FL) and N epsilon-carboxymethyllysine (CML) in a casein-linked preparation. The three different model proteins, casein-LAL, casein-FL and casein-CML were prepared by heating casein either in strong alkaline conditions at 105 degrees C for 1 h, in the presence of glucose at 65 degrees C for 68 h, or in the presence of glyoxylic acid at 37 degrees C for 19 h. Finally, the degree of lysine modification achieved was 39%, 75% and 55% for the casein-LAL, casein-FL and casein-CML, respectively. The calculation of lysine recovery and the respective analysis of each single modified casein (LAL-, FL- and CML-MP) for the selected fortified compound and each other compound vice versa proved that the individual procedure provides a specific fortification for LAL, FL and CML, respectively. The modified proteins are suitable as reference model proteins to be investigated for specific biological and toxicological effects of casein-linked LAL, FL and CML.
Caseins↗
[The effect of enzymatic modification on lysinoalanine formation in field-bean protein isolate and beta-casein].
Lysinoalanine (LAL) was determined in alkali-treated partial hydrolysates (PH) of casein, peptides isolated from these PH and in PH of field-bean protein to clarify whether intermolecular or intramolecular LAL bridges are preferentially formed. Furthermore, the formation of LAS in plasteins was studied as a contribution to plastein research. The formation of LAL in the peptide mixtures of beta-casein and the decrease of the LAL content in the PH (as compared to intact proteins) indicates that the formation of LAL favours the intramolecular cross-linking of polypeptide chains. The LAL content decreases as the degree of hydrolysis of the PH of the field-bean protein isolate increases, and depends upon the protease used in the production of the hydrolysates. The LAL contents of the alkali-treated plasteins are less than those of the initial hydrolysates. The decrease of the LAL content is directly proportional to the hydrolysis proceeding during the plastein reaction.
Amino Acids↗
Dose-dependent utilisation of casein-linked lysinoalanine, N(epsilon)-fructoselysine and N(epsilon)-carboxymethyllysine in rats.
During the heat treatment of protein-containing foods, the amino acid lysine is most prone to undergo chemical reactions in the course of amino acid cross-linking or Maillard reactions. Among the reaction products formed, lysinoalanine (LAL), N(epsilon)-fructoselysine (FL) and N(epsilon)-carboxymethyllysine (CML) are those which serve as sensitive markers for the heat treatment applied. From a nutritional perspective, these compounds are ingested with the diet in considerable amounts but information about their metabolic transit and putative in vivo effects is scarce. In the present study, casein-linked LAL, FL and CML were administered to rats in two different doses for 10 days. Quantitation of LAL, FL and CML in plasma, tissue and faeces samples revealed that the kidneys are the predominant sites of accumulation and excretion. The maximum percent of dietary LAL, FL and CML excreted in the urine was 5.6, 5.2 and 29%, whereas the respective recoveries in the kidneys were 0.02, 26 and 1.4%. The plasma and tissue analyses revealed that the endogenous load of either compound is increased by its dietary intake. But the dose-dependent utilisation of dietary protein-linked LAL, FL and CML in rats has been demonstrated for the first time to vary substantially from each other.
Animals↗
Effect of protein treatment on the enzymatic hydrolysis of lysinoalanine and other amino acids.
Lysinoalanine (LAL) release from alkali-treated proteins by proteolytic enzymes could be measured on a large scale using an in vitro digestion method based on a two-step hydrolysis with pepsin and pancreatin. The hydrolysis was carried out in a dialysis bag, and the digestion products collected and analyzed. Applying this procedure to alkali-treated soybean and rapeseed protein showed that the release of LAL was not related to its concentration in the protein. Instead, LAL release depended on the nature of the protein, the length of the treatment, and the presence of untreated protein. The enzymatic procedure was also used to measure the release of other amino acids in treated and untreated proteins. This in vitro method could directly measure beneficial and adverse effects of processing on amino acid digestibility.
Alkalies↗