TOPS Enzyme Spectrophotometric Determination of Free Fatty Acids
TOPS is one of the new Trinder’s reagents, with high sensitivity, good specificity, and low interference. It is suitable for the detection of a variety of biochemical indicators, such as the TOPS enzymatic photometric determination of serum free fatty acid content in the kit.
Free fatty acid, the non-esterified fatty acid NEFA, is a hydrolysate of human body fat, reflecting the body's fat metabolism and blood lipid level. If its content is too high or too low, it means that the body may have symptoms. At present, the detection of free fatty acids is mainly through kits, usually by enzymatic determination. The reaction principle is mostly based on the Trinder reaction mode. The specific reaction process is as follows:
The free fatty acid (NEFA) in the serum sample reacts with ATP and coenzyme A (CoA) under the catalysis of fatty acyl-CoA synthase to generate fatty acyl-CoA (Acyl-CoA), AMP and phosphate (PPi). Acyl-CoA is oxidized into Enoyl-CoA (Enoyl-CoA) and hydrogen peroxide under the catalysis of fatty acyl-CoA oxidase, and finally hydrogen peroxide is catalyzed by peroxidase with 4-aminoantibi Lin reacts with TOPS to form a red quinone imine compound.
TOPS chromogen substrate
Optimization of TOPS method for determination of free fatty acids:
During the reaction, H2O2 will react with the sulfhydryl group on Coenzyme A to consume part of H2O2, which will lead to a reduction in color products, and the measured free fatty acid concentration will be correspondingly reduced. Improve the optimization method:
1.The solution can be solved by adding N-ethylmaleimide, but the reagent is unstable and will react with fatty acyl-CoA oxidase.
2.Add metal ions (one or more of Pb, Ag, Hg, Cr, such as Pb(NO3)2, AgNO3), so that the metal ions can undergo ion exchange and coordination reactions with the sulfhydryl group on Coenzyme A to form a stable Chelate, eliminate the effect of sulfhydryl group on coenzyme A on hydrogen peroxide production. Thus, the purpose of improving the accuracy of the free fatty acid detection kit and increasing the stability of the kit is achieved.
Use Trinder's chromogen to determine free fatty acids. In addition to TOPS, chromogen substrates such as TOOS and ADPS can also be used. There are ten kinds of chromogen substrates developed and produced by Desheng, including TOOS, TOPS, MAOS, etc., as well as supporting Buffer and enzyme preparations.
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