Indicator amino acid oxidation technique

Another method of determining protein requirements is the indicator amino acid oxidation technique. This method utilizes a carbon-labeled isotope (L-[1- C]) tracer that is ingested orally, and oxidation of this labeled carbon is measured in expired breath as COa. This method is based on the assumption that if one indispensable amino acid is dehcient, all other amino acids will be oxidized until that particular indispensable amino acid is available in adequate amounts, at which point oxidation of the amino acid pool, including the tracer, will be the lowest [32]. Using the indicator amino acid technique, researchers reported that protein recommendations are as much as 30 % higher than the WHO recommendations that are based on nitrogen balance studies [33]. The indicator amino acid oxidation method may help researchers reevaluate current protein requirements [34, 35]. 

Humayim MA, Elango R, BaU RO, Pencharz PB. Reevaluation of the protein requirement in young men with the indicator amino acid oxidation technique. Am J CUn Nutr. 2007 86(4) 995-1002. 

Kurpad AV, Raj T, Regan MM, Vasudevan J, Caszo B, Nazareth D, Gnanou J, Young VR. Threonine requirements of healthy Indian men, measured by a 24-h indicator amino acid oxidation and balance technique. Am J Clin Nutr 2002 76 789-797. 

Electronic absorption spectroscopy can be used to determine the general structure of porphyins and their derivatives. The oxidation and coordination state of the iron and the identity of the amino acid that ligates the heme can be examined by comparing the absorption spectrum of the protein of interest with the spectra of known heme proteins (45, 65). General characterization with electronic absorption spectroscopy indicated that NO and CO, but not O2, bind to the sGC heme moiety (66). Dynamic studies of ligand binding and dissociation also can be examined with this technique on psec-msec time scales with standard stopped-flow systems. 

The most popular method involves 2-thiobarbituric acid (TBA) two molecules of 2-thiobarbituric acid are condensed with malonaldehyde. The emergent chromogen — the two tautomeric structures of the red TBA-malonaldehyde adduct — is determined at 532 nm, and also often at 450 nm, to determine aUcenals and aUcanals, respectively. The qualitative Kreis test was based on a similar principle it involved detection of the epihydrine aldehyde — a tautomeric malondialdehyde — in a color reaction with resorcine or phloroglucinol. The popularity of the TBA test stems from a correlation between the results and sensory evaluations. Paradoxically, this is related to the most important drawback of the TBA technique — its lack of specificity. In addition to the reaction with malonaldehyde, TBA forms compounds of identical color with other aldehydes and ketones, products of aldehyde interaction with nitrogen compounds, and also with saccharides, ascorbic acid, creatine, creatinine, trimethylamine oxide, trimethylamine, proteins, and amino acids. For this reason, the TBA test may even be treated as a proteolysis indicator (Kolakowska and Deutry, 1983). Recently, TBA-reactive substances (TEARS) were introduced, primarily to stress that the reaction involves hydroperoxides in addition to aldehydes. Due to the nonspecificity of the TEARS test, its results reflect the rancidity of food better than other conventional methods, especially off-flavor, which is caused by volatiles from lipids as well as being affected by products of lipids interaction with nitrogenous compounds. 

PDA deteetion is now a mature technique, with nearly 20 years of practical application in the laboratory. The analysis and quantitation of aromatic amino acids in peptides and proteins are probably one the most widely reported uses of PDA for these molecules. Conformational and stability analyses of proteins are another significant application. Also useful, although less frequently appearing in the literature, is PDA deteetion of a variety of naturally occurring chromophores and detection of chemical modifications such as oxidation that result in spectral changes. In all likelihood, chromophore analysis is more widespread than the literature indicates and is just routinely applied with little fanfare. 

Despite the extensive use to which photochemical oxidations of proteins have been put very little attention has been given to identifying the end products of the photoreactions. Apart from methionine which is converted to methionine sulfoxide and more slowly to its sulfone, the fate of the other amino acid residues of irradiated proteins remains largely unknown. On the basis of limited chemical studies, it is clear, however, that photoreactions usually lead to a mixture of products. The multiplicity of products formed by photooxidation of enzymes will undoubtedly limit the utility of the technique. There are in fact numerous examples from work dealing with the chemical modification of enzymes which indicate that modification of a particular residue with different chemical reagents leads to enzyme derivatives with different biological and physico-chemical properties. 

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