Cocktail method for detecting activities of UGT (uridine diphosphoglucuronyl transferase) enzymes in liver microsome

A liver microsome and enzyme activity technology, applied in the field of medicine, can solve problems that are difficult to meet the requirements of fast, accurate, convenient and economical

Active Publication Date: 2017-05-31
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional single-probe drug research method can only reflect the activity of one enzyme in a single ex...

Method used

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  • Cocktail method for detecting activities of UGT (uridine diphosphoglucuronyl transferase) enzymes in liver microsome
  • Cocktail method for detecting activities of UGT (uridine diphosphoglucuronyl transferase) enzymes in liver microsome
  • Cocktail method for detecting activities of UGT (uridine diphosphoglucuronyl transferase) enzymes in liver microsome

Examples

Experimental program
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Effect test

Embodiment 1

[0072] Example 1: Establishing a cocktail method for detecting the enzyme activities of five Ugt subtypes in rat liver microsomes

[0073] (1) Substrate selection of five Ugt subtype enzymes

[0074] The substrate of Ugt1a1 is estradiol, the substrate of UGT1a3 is chenodeoxycholic acid, the substrate of UGT1a6 is serotonin, the substrate of UGT1a9 is propofol, AZTG (the specific subtype is still under study Middle) substrate selection is zidovudine, select the 1 / 5Km-1 / 2Km or lower of the respective substrate concentration for incubation.

[0075] (2) Incubation conditions

[0076] The incubation system mainly consists of 0.5 mg / ml (protein concentration) of rat liver microsomes, 25 μg / ml of alamethicin, 50 mM Tris-HCl (pH 7.4), 5 mM of magnesium chloride and various probe substrates (UGT substrate )composition. First, put the rat liver microsomes, alamethicin, Tris-HCl, magnesium chloride and the probe substrate (substrate of the enzyme) on ice for 15 minutes, so that alame...

Embodiment 2

[0077] Example 2 Establishing a cocktail method for detecting the enzyme activity of five UGT subtypes in human liver microsomes

[0078] Substrate selection, incubation conditions and operating procedures were the same as in Example 1, except that the protein concentration was changed to 0.25 mg / ml, and the incubation time was 60 min. The substrate concentrations were estradiol, 2 μM; chenodeoxycholic acid, 5 μM; serotonin, 500 μM; propofol, 2 μM; zidovudine, 300 μM.

Embodiment 3

[0079] Interaction when five kinds of substrates of embodiment 3 are incubated together

[0080] In rat liver microsomes, the five substrates were mixed together (substrate final concentrations: estradiol, 2 μM; chenodeoxycholic acid, 2 μM; serotonin, 20 μM; propofol, 2 μM; Vudine, 200μM), and co-incubated them to obtain the cocktail result of co-incubation of 5 substrates, compared with the enzyme activity determined by each substrate incubated separately, and intuitively obtained the difference between the cocktail method and the individual incubation, such as figure 1 As shown in A, the ratio of Ugt1a1 in the incubation with the cocktail method is close to 250%, that is, it is greatly affected by the interaction between the substrates, and the comparison between the co-incubation with other substrates and the incubation alone is close to 100%, that is, it is affected by the substrate The interaction between them is small.

[0081] In human liver microsomes, five substrates...

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Abstract

The invention discloses a cocktail method for detecting activities of five UGT enzymes. The method comprises the following steps: selecting substrates with high specificity to the five UGT enzymes, setting a proper concentration, performing substrate interaction survey, mixing and incubating substrates of enzymes UGT1A1/Ugt1a1 and UGT1A6/Ugt1a6 with small interactions, mixing and incubating substrates of UGT1A3/Ugt1a3, UGT1A9/Ugt1a9 and UGT2B7/AZTG, and finally measuring the activities of the UGT enzymes. The cocktail method for detecting the activities of the five UGT enzymes disclosed by the invention can be used for detecting the activities of UGT enzymes in rat and human liver microsome, has the advantages of high efficiency, rapidness and comprehensiveness and can be used for rapidly evaluating the influences of compounds on the activities of UGT enzymes.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a cocktail method for detecting five kinds of UGT enzyme activities in human or rat liver microsomes. Background technique [0002] Uridine diphosphate glucuronosyltransferase (UGT) is the main two-phase metabolic enzyme (including uridine diphosphate glucuronosyltransferase, N-acetyltransferase, methyltransferase, glutathione transferase, Transsulfurase, etc.), mainly located in the smooth endoplasmic reticulum in the liver, its main function is to bind glucuronide to the substrate, making it more polar and easy to excrete, so as to achieve the role of metabolism and detoxification. UGT enzymes not only play an important role in drug metabolism, which can metabolize 35% of drugs (through two-phase metabolism), but also play a crucial role in metabolizing endogenous substances, such as bile acids, bilirubin, steroid hormones, etc. important role. When the activity ...

Claims

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Application Information

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IPC IPC(8): C12Q1/48
CPCC12Q1/48
Inventor 王昕周小静陈昂刘明耀
Owner EAST CHINA NORMAL UNIV
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