Optimization of TOPS Method for Determination of Free Fatty Acids
The determination of free fatty acids in serum or plasma usually adopts Trinder chromogen enzyme photometric method. However, due to the characteristics of this test, the detection of fatty acids is affected by many factors, and the detection method needs to be optimized and improved to improve the accuracy of the detection.
Trinder chromogen detection FFA principle:
The detection method has three reaction steps: free fatty acids (FFA or NEFA) react with excess CoA to generate acyl-CoA under the action of acetyl-CoA synthase (ACS), and react with oxygen under the action of acetyl-CoA oxidase (ACO). The reaction generates 2,3-trans-enoyl CoA and hydrogen peroxide, and the Trinder reaction is used to detect hydrogen peroxide, and the content of FFA can be obtained.
TOPS is recommended for FFA determination of chromogen
Problems in FFA determination and optimization methods:
1. Excessive Coenzyme A in the reaction will interfere with the reaction of hydrogen peroxide and Trinder chromogen, making the whole test result low. N-ethylmaleimide (NEM) can be used to remove excess Coenzyme A. The stability of NEM is very affected. Limited, only one week.
2. The acetyl CoA synthetase and acetyl CoA oxidase used have different substrate specificities for different free fatty acids, causing differences in the determination results. Different enzyme sources have different substrate specificities for free fatty acids with different C chain lengths. That is, the response capacity is different. There are 6 kinds of FFA in human serum, and only enzymes with high specificity to them can make no difference in the measurement results.
3. Due to the influence of CoA on the reaction, the linear range of the data results is narrow. The color of FFA measured on the market is MEHA, TBHB, TOOS, etc., but it is greatly interfered by CoA, and it must be excessive, so interference is required. Less chromogen. SCEP is not interfered by Coenzyme A but is unstable. ADOS and TOPS are less interfered by CoA, and TOPS has a higher molar absorption coefficient, so it is a better chromogen substrate.
4. Add sulfhydryl complex DTNB and MIT to reduce the amount of NEM. The sulfhydryl group of DTNM can react with the sulfhydryl group of CoA to remove the interference to chromogen. A small amount of MIT can remove the sulfhydryl group of CoA and also acts as a preservative. Based on the use of TOPS chromogen, the interference of CoA can be completely eliminated, and the stability is greatly improved.
If you have higher requirements for FFA determination results, you can choose a better quality kit based on the above points. Desheng produces the raw materials for the FFA and other index determination kits. If TOPS is used to determine FFA directly, due to the poor stability of the solution, it is recommended to prepare a working solution for immediate use before use.
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