Analysis of amino acid metabolites
Analysis of amino acid metabolites
Summary
Amino acids are mainly used in the body to synthesize proteins, peptides and other nitrogenous substances that are unique to the body; on the other hand, they can be decomposed into α-keto acids, amines and carbon dioxide through deamination, transamination, combined deamination or decarboxylation. The α-keto acids generated from amino acid catabolism can be converted into sugars, lipids or re-synthesized into certain non-essential amino acids, or oxidized to carbon dioxide and water through the citric acid cycle, and release energy. Currently, there are four main methods used to analyze amino acid metabolites: glutaminase activity analysis, alanine aminotransferase activity analysis, glutamine content determination and glutamate content determination.
Principle
According to different experimental methods, the corresponding principles are also different:
1. The basic principle of method I glutaminase activity analysis is: Glutaminase (GLS) catalyzes the hydrolysis of glutamine to produce glutamate and ammonia, and plays an important regulatory role in nitrogen metabolism, especially in regulating free ammonia content and urea metabolism. The principle of this enzyme activity assay is to detect the rate of increase of ammonia using an alkaline solution of potassium mercuric iodide (i.e., Naïve's reagent), and the enzyme activity can be calculated. The Naïve's reagent reacts with ammonia to form a brown color complex, the absorbance of which at 420 nm is proportional to the ammonia content in the concentration range of less than 1.75 mg/L (N).
2, Method 2: alanine aminotransferase activity analysis of the basic principle is: alanine aminotransferase (ALT) can catalyze the transfer of amino acids from alanine to a α-KG transamination reaction, this reversible amino acid reaction products for the pyruvate and glutamate, in the amino acid metabolism has an important role. The products of this reversible reaction are pyruvate and glutamate, which are important in amino acid metabolism. ALTase activity is high in mammalian hepatocytes, and when hepatocytes are necrotic, ALT is released into the bloodstream and serum ALT activity is significantly increased. Therefore, ALT is recommended by the World Health Organization as the most sensitive indicator of liver impairment.ALT catalyzes the transamination reaction of alanine and α-KG to produce pyruvate and glutamate; addition of 2,4-dinitrophenylhydrazine not only terminates the above reaction, but also adducts with the carbonyl group of ketoacid to produce phenylhydrazone of pyruvate, which appears reddish brown under alkaline conditions and can be used as a molecule for the production of phenylhydrazones. Benzhydrazone is reddish brown in alkaline condition, the absorbance value can be read at 505 nm and the enzyme activity can be calculated.
Method 3: The basic principle of glutamine content determination is: glutamine (GLN) is an essential amino acid, many tissues contain GLN synthetase, which catalyzes the synthesis of GLN. Under stress conditions such as strenuous exercise, trauma, infection, etc., the body's need for GLN may exceed its ability to synthesize GLN. Insufficient synthesis of GLN can lead to reduced protein synthesis and atrophy of the small intestinal mucosa. Therefore, GLN is called a conditionally essential amino acid. Glutamine is formed into glutamic acid and NH by the action of glutaminase, and the amount of glutamine is calculated by measuring the amount of NH.
Method 4: The basic principle of the determination of glutamate content is: Although glutamate is not an essential amino acid in the human body, it has a high nutritional value. After glutamic acid is absorbed into the blood, it is easy to form glutamine with blood ammonia to release the toxic effect of ammonia in the process of metabolism, thus preventing and treating hepatic encephalopathy. As the main energy source of the nerve center and cerebral cortex, glutamate is of great significance in the function of the nervous system. In the presence of NAD＊, aminotransferase and glutamate dehydrogenase catalyze the formation of α-KG, NADH and NH from glutamate, and the amount of NADH detected reflects the amount of glutamate. The amount of intracellular glutamate is related to the amount of the cell's own enzymes and the amount of glutamine in the culture medium. If the assay result is low, it is necessary to increase the concentration of the sample for the assay.
Operation method
Analysis of alanine aminotransferase activity
Principle
The basic principle of alanine aminotransferase activity analysis is: ALT catalyzes the transamination reaction of alanine and α-KG to produce pyruvate and glutamate; addition of 2,4-dinitrophenylhydrazine solution not only terminates the above reaction, but also adducts with the carbonyl group in the ketoacid to produce phenylhydrazone of pyruvate; phenylhydrazone has a reddish-brown color under alkaline conditions, and the absorbance value can be read at 505 nm and the enzyme activity can be calculated. The absorbance value can be read at 505 nm and the enzyme activity can be calculated.
Materials and Instruments
① Sample
② 2,4-Dinitrophenylhydrazine solution
Move
The basic steps of the experiment for alanine transaminase Hoxing analysis are shown below:
A, Addition of 2,4-dinitrophenylhydrazine solution terminates the ALT-catalyzed transamination reaction of alanine and α-KG and adducts with the carbonyl group in keto acids to produce phenylhydrazone pyruvate;
B. Phenylhydrazone is reddish brown under alkaline conditions and the absorbance was read at 505 nm and the enzyme activity was calculated.
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