Tryptophan amino acids liberated from

Introduction of microbiological methods for the determination of amino acids made possible the estimation of the amount of both free and combined amino acids in urine. Dunn et al. (D4), Thompson and Kirby (Tl), Eckhard and Davidson (El), and Woodson et al. (W3) estimated the amount of amino acids liberated in the course of acid or, as in the case of tryptophan determination, alkaline hydrolysis. Microbiological and colorimetric methods used for the determination of certain amino acids present very little opportunity for evaluating the proper quantitative relations between free and combined amino acids, since under the applied condition both combined and free amino acids are equally involved in the reaction. In 1949 Albanese et al. (A3) applied such methods to the quantitative determination of free and combined amino acids in the nondiffusible fraction of urine, and subjected the procedures to broad criticism from just this point of view. 

Metabolic Transit. Free Amadori Compounds. It is well known that the synthetic Amadori compounds of the free amino acids are absorbed by the intestine and excreted unchanged in the urine (9,28,30). The transport is not active as observed with deoxyfructosyltryptophan (30) and c-deoxyfructosyllysine (40), and the level of absorption depends on the nature of the amino acid and on the conditions of ingestion. Nutritional assays and metabolic transit studies performed with radioactive Amadori compounds of tryptophan (12,30), leucine (12), and lysine (9,28,41) given orally or intravenously on normal or anti-biotics-treated animals have shown that the intestinal microflora can regenerate part of the amino acid. This can be absorbed subsequently at a very low level by the caecum or the large intestine and incorporated into the tissue proteins or utilized by the intestinal microflora. Barbiroli (13) showed also that some intestinal enzymes were able to liberate some amino acids from their Amadori compounds but to a very small... 

As has been stated, tryptophan is completely destroyed by acid hydrolysis. Some methods protect tryptophan with compounds such as 2-mercaptoethanol, but these methods do not always give reproducible results. Other methods for the liberation of tryptophan have included proteolysis by enzymes. An early method developed by Spies and Chambers (1949) did not require freed tryptophan. It is a colorimetric assay, reacting the tryptophan with p-dimethylaminobenzaldehyde (PDBA). This method has been useful in pure proteins, but many food matrices have interfering substances. In addition, tryptophan is one of only two amino acids with a strong extinction coefficient in a usable ultraviolet (UV) range (approximately 280 nm depending on solvent, etc.). However, the most reliable method of tryptophan analysis is to release the amino acid from protein with alkaline hydrolysis (Lucas et al., 1980) and then use chromatography for quantification. 

M hydrochloric acid at 107°C for 18 hr. The hydrolysate contained tryptophan, valine, glycine, proline, threonine, aspartic acid, and histidine in the (nearest integer) ratio of 1 1 1 1 2 2 1. The amino-terminal residue of the flavin peptide was found to be aspartic acid by the subtractive Edman method, with glycine as the next residue. Hydrolysis of the flavin peptide with 6 M hydrochloric acid for 17 hr at 95°C gave a histidylflavin which was purified by thin layer chromatography. Histidine was liberated from this histidylflavin by incubation at 125°C in 6 M hydrochloric acid. 

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