CN109182451B - Novel specific probe reaction of cytochrome CYP3A7 enzyme and application thereof - Google Patents
Novel specific probe reaction of cytochrome CYP3A7 enzyme and application thereof Download PDFInfo
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Abstract
The invention provides a novel specific probe reaction of cytochrome CYP3A7 enzyme and application thereof: the 19-hydroxylation reaction of deoxycholyl glycine and the 19-hydroxylation reaction of deoxycholyl taurine are used as probe reactions of CYP3A7 enzyme to detect the activity of the enzyme; the method takes deoxycholic glycine or deoxycholic taurine as a probe substrate, and can realize the quantitative detection of the CYP3A7 enzyme activity in biological samples from different sources by quantitatively measuring the generation rate of a specific reaction product, namely 19-hydroxydeoxycholic glycine or 19-hydroxydeoxycholic taurine, in unit time as an evaluation index of the cytochrome CYP3A7 enzyme activity.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a specific probe reaction of deoxycholinylglycine and deoxycholinyltaurine serving as cytochrome CYP3A7 enzyme and application thereof.
Background
Cytochrome P450 enzymes (CYPs) are a superfamily of heme-thiolated proteins that are widely involved in the metabolic conversion and elimination of endogenous steroids and exogenous substances including drugs and environmental compounds. According to the degree of homology of amino acid sequences, the members of CYPs are divided into three classes of family, subfamily and subtype enzymes in turn. The subfamilies of CYP1A, CYP2A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A are the main I-phase drug metabolizing enzymes in human bodies, are mainly expressed in the liver, and participate in the metabolic clearance of about 75% of marketed drugs. Among them, the CYP3A subfamily is the most important, and the expression level in liver and intestinal tract is 40% and 80% of total CYP, respectively, and is involved in the metabolic clearance of about 46% of marketed drugs (Nat Rev Drug Discov. 2005, 4(10): 825-833). The expression level, catalytic function and metabolic contribution rate of CYP3A enzyme are greatly different from individual to individual due to genetic factors, non-genetic factors and drug interactions, which results in a great difference in the in vivo drug concentration levels after different individuals use the same drug treatment regimen, thereby leading to distinct safety and efficacy outcomes (Annu Rev Pharmacol Toxicol. 1998, 38: 389-430). Therefore, the design of sensitive and effective CYP3A enzyme activity detection methods is extremely urgent, which has important significance for drug screening in the drug development stage and individualized treatment in the drug application stage.
The human body mainly expresses three CYP3A subtype enzymes, namely CYP3A4, CYP3A5 and CYP3A7, and the protein sequences of the three enzymes are more than 82% consistent, but the enzymes show remarkably different substrate recognition, metabolic reaction catalytic activity and regioselectivity (Annu Rev Pharmacol Toxicol 1999, 39: 1-17). CYP3a4 and CYP3a5 are major CYP3A subtype enzymes that exert drug metabolism functions in adult humans and have highly similar substrate recognition. CYP3a7 is the major CYP3A subtype enzyme in newborns and infants: CYP3A7 accounts for about 30% of total CYP expression in the neonatal period (within 1 week after birth), when CYP3A4 is expressed at a level much lower than that of adults; in later infancy and childhood, CYP3A7 expression is gradually down-regulated, whereas CYP3A4 expression is gradually up-regulated until adolescence develops to adult levels (Clin Pharmacokinet 1999, 37(6): 485-). 505). The CYP3a7 expression level is generally low in adults, but CYP3a7 x 1C gene carriers have some expression. Studies have shown that the CYP3A7 x 1C gene is involved in the pathogenesis of polycystic ovary syndrome (J Clin Endocrinol Metab. 2008, 93: 2909-12), in the outcome of chemotherapy failure in chronic lymphocytic leukemia, breast and lung Cancer (Cancer Res. 2016, 76(6): 1485-93), and in the degree of fetal development during pregnancy (Hum Mol Genet. 2018, 27(4): 742-56). Therefore, the design of a sensitive and effective CYP3A7 enzyme activity detection method has important significance for the development and application of pediatric drugs, has potential important value for the auxiliary diagnosis of metabolic diseases related to CYP3A7, the development of pregnant fetuses and the evaluation of chemotherapy prognosis of tumor patients, and can be developed into a key screening technology of tumor-targeted therapeutic drugs for specifically inhibiting CYP3A7 activity (Biochim Biophys acta. 2011, 1814(1): 161-.
The In vitro evaluation system of CYP3A enzyme activity mainly comprises recombinant single enzyme, mammalian subcellular components, freshly isolated liver cells, liver slices, liver perfusion and the like (Toxicol In vitro 2006, 20(2):135-53), wherein the commercialized recombinant cytochrome CYP3A7 single enzyme mainly comes from a clone expression system established by bacteria, insect cells, mammalian cells and yeasts, and the subcellular components mainly comprise microsomes, cytoplasm and S9 components. By using the standardized evaluation systems, and combining corresponding cofactors (such as NADPH and the like) and incubation conditions, the CYP3A7 enzyme activity expressed in the various systems can be characterized by detecting the substrate metabolic clearance rate or the product generation rate of the probe reaction.
At present, the activity of CYP3A enzyme is mainly determined by using a specific probe substrate and a probe reaction thereof (Drug Metab Rev. 2007, 39(4): 699-721). Probes that have been found or have been widely used in vitro and in vivo studies are specific for CYP3a4 and/or CYP3a5, such as Midazolam 1'-hydroxylation (Midazolam 1' -hydroxylation), Testosterone 6beta-hydroxylation (Testosterone 6beta-hydroxylation), Gomisin A8-hydroxylation (gomissin A8-hydroxylation), Cortisol 6beta-hydroxylation (Cortisol 6beta-hydroxylation), Cholesterol 4beta-hydroxylation (Cholesterol 4beta-hydroxylation), and the like. It is considered that 16-hydroxylation (Dehydroepiandrosterone 16alpha-hydroxylation) of Dehydroepiandrosterone and 2alpha-hydroxylation (Testosterone 2alpha-hydroxylation) of Testosterone can be used as specific probe reactions of CYP3A7, but the results of recombinant single enzyme test show that the specificity of the two probe reactions is far from meeting the requirements of practical application: CYP3A7 has only 3.0 times the catalytic activity of CYP3A4 for dehydroepiandrosterone 16alpha-hydroxylation (J Pharmacol Exp ther. 2003, 307(2):573-82), and CYP3A7 has only 2.5 times the catalytic activity of CYP3A4 for 2alpha-hydroxylation of testosterone (J Pharmacol Exp ther. 2005, 314(2): 626-635). In summary, there is no specific probe reaction for measuring the activity of CYP3a7, but the key to the CYP3a7 activity measuring technique is to select a probe reaction with high specificity.
The inventors of the present invention have disclosed in the patent of invention (201810543953.0) that the 19-hydroxylation reaction of deoxycholic acid is the result of a study of the probe reaction specific to CYP3A 7. Deoxycholic glycine (glycodeoxycholic acid, N- (3 alpha, 12 alpha-Dihydroxy-24-oxochol-24-yl) glycine) and deoxycholic taurine (taurodeoxycholic acid, N- (3 alpha, 12 alpha-Dihydroxy-24-oxochol-24-yl) taurine) are conjugated bile acids in which deoxycholic acid is respectively combined with glycine or taurine, and the 19-hydroxylation reaction of deoxycholic glycine and deoxycholic taurine is the specific probe reaction for detecting cytochrome CYP3A7 enzyme disclosed for the first time in the invention.
Disclosure of Invention
The invention aims to provide a specific probe reaction of cytochrome CYP3A7 enzyme and application thereof, in particular to a 19-hydroxylation reaction of deoxycholic acid glycine and deoxycholic acid taurine, which is a high-specific probe reaction of CYP3A7 and can be used for quantitatively measuring the activity of the CYP3A7 enzyme in biological samples from different sources. Compared with the potential probe reactions reported in the literature (16-hydroxylation reaction of dehydroepiandrosterone and 2alpha-hydroxylation reaction of testosterone), other known CYP subtype enzymes including CYP3A4 and CYP3A5 have no catalytic activity at all for deoxycholylglycine and deoxycholyltaurine-19 hydroxylation reactions, and the reactions can characterize the activity of CYP3A7 enzyme with high specificity.
The invention specifically provides a novel specific probe reaction of cytochrome CYP3A7 enzyme, which is characterized in that: the substrate of the probe reaction is deoxycholic glycine or deoxycholic taurine, and the structure of the substrate is shown as formula (1) or (2) in figure 1; the products of the probe reaction are respectively 19-hydroxydeoxycholic glycine or 19-hydroxydeoxycholic taurine, and the structures of the products are shown as formulas (3) or (4) in figure 1.
The invention also provides a method for quantitatively determining the activity of CYP3A7 enzyme in an in vitro biological sample by using the specific probe reaction, wherein deoxycholyl glycine or deoxycholyl taurine is used as a substrate of the probe reaction, and the activity of the CYP3A7 enzyme in biological samples and cells of different sources is determined by quantitatively detecting the generation amount of 19-hydroxylation products in unit time, and the specific determination method comprises the following steps:
in the system, deoxycholyl glycine or deoxycholyl taurine is used as a substrate for reaction, and the concentration range of the substrate is 1-500 mu M;
in an incubation system with pH7.4 and a cofactor NADPH or a generation system thereof, the reaction temperature is between 10 and 60 ℃;
the reaction time is 0-720 min, and the reaction is stopped when the generated 19-hydroxydeoxycholic glycine or 19-hydroxydeoxycholic taurine reaches the detection and quantification limit and the substrate conversion rate is lower than 20%;
detecting the amount of 19-hydroxylation product produced in unit time, and determining the rate of production of reaction products, thereby achieving detection of cytochrome CYP3A7 enzyme activity.
The application of the specific probe reaction provided by the invention is suitable for detecting the activity of CYP3A7 enzyme in preparations such as cells, microsomes, cytoplasm, S9 components and the like from human tissues and organs, and is particularly suitable for calibrating the activity of CYP3A7 recombinase produced by a clone expression system established by bacteria, insect cells, mammalian cells and yeasts.
Sample processing methods in which probe reaction products are detected include direct processing methods, processing methods in which the products are hydrolyzed to an unbound form (e.g., using acid hydrolysis), and processing methods in which the products are enzymatically hydrolyzed to an unbound form (e.g., using cholyl glycinate hydrolase). The detection method comprises the detection of ultraviolet absorption spectrum, mass spectrum, radioactive isotope tracing technology, immunoassay technology, fluorescence excitation spectrum, evaporative light scattering or electrochemical spectrum; the detector comprises one or more of ultraviolet absorption spectrum detector, mass spectrum, radioactive isotope tracing technology, evaporative light scattering or electrochemical spectrum detector connected in series.
Deoxycholic glycine or deoxycholic taurine can generate a plurality of hydroxylated metabolites through the metabolism of cytochrome CYP3A7, 19-hydroxydeoxycholic acid (Chem Pharm Bull. 1995, 43(9): 1551-. The examination by using cytochrome CYP recombinase and a human liver microsome incubation system proves that the 19-hydroxylation reaction of deoxycholic acid or deoxycholic acid taurine is selectively catalyzed only by CYP3A7, and the other CYP subtype enzymes comprise CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9 x 1, CYP2C18, CYP2C19, CYP2D 61, CYP3A4, CYP3A5, CYP2E1, CYP2J2, CYP4A11, CYP4F2, CYP4F3B and CYP4F12 cannot catalyze the 19-hydroxylation reaction of deoxycholic acid or deoxycholic acid taurine (as shown in figures 2 and 3). By examining the production rate curve of 19-hydroxylation product during the 12-hour incubation of deoxycholic glycine or deoxycholic taurine in the CYP3A4, CYP3A5 and CYP3A7 recombinase systems, it was confirmed that the 19-hydroxylation reaction of deoxycholic glycine or deoxycholic taurine was selectively catalyzed only by CYP3A7, while CYP3A4 and CYP3A5 were not catalyzed (as shown in FIGS. 4 and 5). Through observing the enzyme reaction kinetics of deoxycholylglycine (1-300 mu M) catalyzed by CYP3A4 and CYP3A7 recombinase (shown in figure 6), it is proved that CYP3A4 can not catalyze the 19-hydroxylation reaction of the deoxycholylglycine in the test substrate concentration range, the 19-hydroxylation reaction rate catalyzed by CYP3A7 conforms to the classic Michaelis kinetic equation, and the maximum reaction rate (Vmax) and the Michaelis constant (Km) are 715.1pmol/min/nmol P450 and 110.5 mu M respectively; similarly, for the enzyme kinetics of deoxycholyl taurine (as shown in fig. 7), CYP3a4 was unable to catalyze the 19-hydroxylation reaction of deoxycholyl taurine over the range of substrate concentrations tested (1-300 μ M), whereas the rate of the 19-hydroxylation reaction catalyzed by CYP3a7 was in accordance with the classical mie kinetic equation, with the maximum reaction rate (Vmax) and the mie constant (Km) of 2035pmol/min/nmol P450 and 151 μ M, respectively.
The in vitro activity of the cytochrome CYP3A7 enzyme detected by the cytochrome CYP3A7 specific probe reaction has the following outstanding advantages: (1) high specificity: deoxycholic glycine or deoxycholic taurine can be metabolized into 19-hydroxylation products by cytochrome CYP3A7 with high specificity; (2) substrate accessibility: deoxycholic glycine and deoxycholic taurine are derived from bile of various mammals, have high content, and can be directly extracted and separated or chemically synthesized.
Drawings
FIG. 1 shows a 19-hydroxylation reaction pathway of deoxycholyl glycine and deoxycholyl taurine catalyzed by cytochrome CYP3A 7.
FIG. 2 shows the results of screening experiments for human cytochrome CYP recombinase in deoxycholic glycine 19-hydroxylation reaction.
FIG. 3 shows the results of screening test of human cytochrome CYP recombinase by the 19-hydroxylation reaction of deoxycholyltaurine.
FIG. 4 is a graph of the production rate of 19-hydroxylation product during incubation of deoxycholylglycine (50. mu.M) in the cytochrome CYP3A4, CYP3A5, and CYP3A7 recombinase system for 12 hours.
FIG. 5 is a graph showing the production rate of 19-hydroxylation product during incubation of deoxycholyltaurine (50. mu.M) in a cytochrome CYP3A4, CYP3A5 and CYP3A7 recombinase system for 12 hours.
FIG. 6 is a graph showing the enzyme kinetics of deoxycholylglycine 19-hydroxylation reaction in cytochrome CYP3A4 and CYP3A7 recombinant enzyme systems.
FIG. 7 is an enzyme kinetic curve of deoxycholinesterase 19-hydroxylation reaction in cytochrome CYP3A4 and CYP3A7 recombinant enzyme systems.
FIG. 8 is a liquid chromatography-mass spectrometry detection chromatogram after deoxycholic glycine and 19-hydroxydeoxycholic glycine are subjected to enzymolysis treatment to obtain an unbound form in a reaction system of a mixed human liver microsome, a cytochrome CYP3A7 recombinase, a cytochrome CYP3A5 recombinase and a cytochrome CYP3A4 recombinase.
FIG. 9 is a liquid chromatography-mass spectrometry detection chromatogram obtained by subjecting deoxycholyl taurine and 19-hydroxydeoxycholyl taurine to enzymatic hydrolysis treatment to obtain an unbound form in a reaction system of a mixed human liver microsome, a cytochrome CYP3A7 recombinase, a cytochrome CYP3A5 recombinase and a cytochrome CYP3A4 recombinase.
FIG. 10 shows the 19-hydroxylation reaction rate of deoxycholylglycine in liver microsomes of adult human individuals.
FIG. 11 shows the 19-hydroxylation reaction rate of deoxycholyltaurine in liver microsomes of adult individuals.
Detailed Description
The following examples are intended to further illustrate the invention, but are not intended to limit its scope.
Example 1
Use of deoxycholylglycine 19-hydroxylation reaction for screening time-independent CYP3A7 enzyme inhibitors
A commercial adult mixed liver microsome and a time-independent inhibitor of each CYP enzyme are purchased, and the inhibition effect of each inhibitor on the CYP3A7 enzyme activity is tested by using the 19-hydroxylation reaction of deoxycholic glycine, and the specific operation flow is as follows:
(1) the reaction starting concentration of deoxycholyl glycine in the 100 mu L in-vitro reaction system is 50 mu M, and contains: 10 mM glucose-6-phosphate, 1 unit glucose-6-phosphate dehydrogenase, 4 mM MgCl 21 mM NADP + and a certain concentration of CYP enzyme inhibitor (alpha-naphthol 0.1 mu M, tranylcypromine 0.2 mu M, sertraline 10 mu M, Montelukast 0.5 mu M, quercetin 1 mu M, sulfabenezole 1 mu M, nootkatone 1 mu M, quinidine 1 mu M, diethyl dithiocarbamate 20 mu M, fluconazole 10 mu M or ketoconazole 0.5 mu M), pre-incubating for 5 minutes under the condition of 37 ℃ C;
(2) adding liver microsomes (the concentration of microsomes protein in the system is 0.5 mg/mL) into the reaction system to start reaction;
(3) after reacting for 60 minutes, adding 300 mu L of acetonitrile to terminate the reaction;
(4) centrifuging for 10 minutes at 12,000 × g using a high-speed refrigerated centrifuge;
(5) taking 100 mu L of centrifuged supernatant, and carrying out vacuum centrifugation and drying at 40 ℃;
(6) adding 150 μ L sodium acetate buffer solution (pH 5.0, containing 100U of cholyl glycine hydrolase) into the residue, shaking and incubating at 37 ℃ for 6 h, and carrying out enzymolysis on the hydroxylation product in a bound form into an unbound form;
(7) adding 800 mu L acetonitrile (containing 1% formic acid) to terminate the enzymolysis reaction;
(8) centrifuging for 10 minutes at 12,000 × g using a high-speed refrigerated centrifuge;
(9) taking 400 mu L of centrifuged supernatant, and carrying out vacuum centrifugation and drying at 40 ℃;
(10) adding 60 μ L acetonitrile and 60 μ L water into the volatilized residue, and shaking to dissolve;
(11) the UPLC-MS/MS is adopted to detect the generation amount of the 19-hydroxydeoxycholic acid, and the result shows that: selective inhibitors of other CYP enzymes, including alpha-naphthol (CYP 1a2 inhibitor), tranylcypromine (CYP 2a6 inhibitor), sertraline (CYP 2B6 inhibitor), montelukast (CYP 2C8 inhibitor), quercetin (CYP 2C8 inhibitor), sulfaphenazole (CYP 2C9 inhibitor), nootkatone (CYP 2C19 inhibitor), quinidine (CYP 2D6 inhibitor), diethyldithiocarbamate (CYP 2E1 inhibitor), fluconazole (CYP 2C/3A inhibitor), have no inhibitory effect on the 19-hydroxylation reaction of deoxycholic glycine; the CYP3A inhibitor ketoconazole has a remarkable inhibiting effect (p < 0.05) on the 19-hydroxylation reaction of deoxycholylglycine, and the 0.5 mu M ketoconazole can reduce the generation amount of 19-hydroxyl products by 56%.
Example 2
Application of deoxycholic acid taurine 19-hydroxylation reaction in screening time-independent CYP3A7 enzyme inhibitor
The method is characterized in that a commercial adult mixed liver microsome and a time-independent inhibitor of each CYP enzyme are purchased, and the inhibition effect of each inhibitor on the CYP3A7 enzyme activity is tested and screened by using the 19-hydroxylation reaction of deoxycholyl taurine, and the specific operation flow is as follows:
(1) the reaction starting concentration of the deoxycholyl taurine in the 100 muL in-vitro reaction system is 50 muM, and the reaction starting concentration comprises: 10 mM glucose-6-phosphate, 1 unit glucose-6-phosphate dehydrogenase, 4 mM MgCl 21 mM NADP + and a certain concentration of CYP enzyme inhibitor (alpha-naphthol 0.1 mu M, tranylcypromine 0.2 mu M, sertraline 10 mu M, Montelukast 0.5 mu M, quercetin 1 mu M, sulfabenezole 1 mu M, nootkatone 1 mu M, quinidine 1 mu M, diethyl dithiocarbamate 20 mu M, fluconazole 10 mu M or ketoconazole 0.5 mu M), pre-incubating for 5 minutes under the condition of 37 ℃ C;
(2) adding liver microsomes (the concentration of microsomes protein in the system is 0.5 mg/mL) into the reaction system to start reaction;
(3) after reacting for 60 minutes, adding 300 mu L of acetonitrile to terminate the reaction;
(4) centrifuging for 10 minutes at 12,000 × g using a high-speed refrigerated centrifuge;
(5) taking 100 mu L of centrifuged supernatant, and carrying out vacuum centrifugation and drying at 40 ℃;
(6) adding 150 μ L sodium acetate buffer solution (pH 5.0, containing 100U of cholyl glycine hydrolase) into the residue, shaking and incubating at 37 ℃ for 6 h, and carrying out enzymolysis on the hydroxylation product in a bound form into an unbound form;
(7) adding 800 mu L acetonitrile (containing 1% formic acid) to terminate the enzymolysis reaction;
(8) centrifuging for 10 minutes at 12,000 × g using a high-speed refrigerated centrifuge;
(9) taking 400 mu L of centrifuged supernatant, and carrying out vacuum centrifugation and drying at 40 ℃;
(10) adding 60 μ L acetonitrile and 60 μ L water into the volatilized residue, and shaking to dissolve;
(11) the UPLC-MS/MS is adopted to detect the generation amount of the 19-hydroxydeoxycholic acid, and the result shows that: other selective inhibitors of CYP enzymes, including alpha-naphthol (CYP 1a2 inhibitor), tranylcypromine (CYP 2a6 inhibitor), sertraline (CYP 2B6 inhibitor), montelukast (CYP 2C8 inhibitor), quercetin (CYP 2C8 inhibitor), sulfaphenazole (CYP 2C9 inhibitor), nootkatone (CYP 2C19 inhibitor), quinidine (CYP 2D6 inhibitor), diethyldithiocarbamate (CYP 2E1 inhibitor), fluconazole (CYP 2C/3A inhibitor), have no inhibitory effect on the 19-hydroxylation reaction of deoxycholtaurine; the CYP3A inhibitor ketoconazole has obvious inhibition effect (p < 0.05) on 19-hydroxylation reaction of deoxycholyl taurine, and 0.5 mu M ketoconazole can reduce the generation amount of 19-hydroxylation products by 53%.
Example 3
19-hydroxylation of deoxycholylglycine for determination of CYP3A7 enzyme Activity in liver microsomes of 14 adult individuals
Commercially available 14 liver microsomes from different adult individuals were purchased, and the activity of CYP3A7 enzyme in human liver microsomes samples was measured by 19-hydroxylation reaction of deoxycholic glycine, according to the following specific procedure:
(1) the reaction starting concentration of deoxycholyl glycine in the 100 mu L in-vitro reaction system is 50 mu M, and contains: 10 mM glucose-6-phosphate, 1 unit glucose-6-phosphate dehydrogenase, 4 mM MgCl2And 1 mM NADP + at 37 ℃Pre-incubation for 5 minutes;
(2) adding liver microsomes (the concentration of microsomes protein in the system is 0.5 mg/mL) into the reaction system to start reaction;
(3) after reacting for 60 minutes, adding 300 mu L of acetonitrile to terminate the reaction;
(4) centrifuging for 10 minutes at 12,000 × g using a high-speed refrigerated centrifuge;
(5) taking 100 mu L of centrifuged supernatant, and carrying out vacuum centrifugation and drying at 40 ℃;
(6) adding 150 μ L sodium acetate buffer solution (pH 5.0, containing 100U of cholyl glycine hydrolase) into the residue, shaking and incubating at 37 ℃ for 6 h, and carrying out enzymolysis on the hydroxylation product in a bound form into an unbound form;
(7) adding 800 mu L acetonitrile (containing 1% formic acid) to terminate the enzymolysis reaction;
(8) centrifuging for 10 minutes at 12,000 × g using a high-speed refrigerated centrifuge;
(9) taking 400 mu L of centrifuged supernatant, and carrying out vacuum centrifugation and drying at 40 ℃;
(10) adding 60 μ L acetonitrile and 60 μ L water into the volatilized residue, and shaking to dissolve;
(11) the UPLC-MS/MS is adopted to detect the generation amount of 19-hydroxydeoxycholic acid, and the result shows that the 19-hydroxylation reaction of deoxycholic acid is used as a probe, the generation rates of 19-hydroxylation products in liver microsomes of adult individuals with different sources are obviously different (figure 10), and the difference between the highest activity and the lowest activity reaches 262.1 times.
Example 4
19-hydroxylation of deoxycholyltaurine for determination of CYP3A7 enzyme Activity in liver microsomes of 14 adult individuals
The activity of CYP3A7 enzyme in human liver microsome samples was determined by 19-hydroxylation of deoxycholyltaurine using commercially available 14 liver microsomes from different adult individuals, according to the following specific procedures:
(1) the reaction starting concentration of the deoxycholyl taurine in the 100 muL in-vitro reaction system is 50 muM, and the reaction starting concentration comprises: 10 mM ofGlucose-6-phosphate, 1 unit of glucose-6-phosphate dehydrogenase, 4 mM MgCl2And 1 mM NADP +, pre-incubated at 37 ℃ for 5 min;
(2) adding liver microsomes (the concentration of microsomes protein in the system is 0.5 mg/mL) into the reaction system to start reaction;
(3) after reacting for 60 minutes, adding 300 mu L of acetonitrile to terminate the reaction;
(4) centrifuging for 10 minutes at 12,000 × g using a high-speed refrigerated centrifuge;
(5) taking 100 mu L of centrifuged supernatant, and carrying out vacuum centrifugation and drying at 40 ℃;
(6) adding 150 μ L sodium acetate buffer solution (pH 5.0, containing 100U of cholyl glycine hydrolase) into the residue, shaking and incubating at 37 ℃ for 6 h, and carrying out enzymolysis on the hydroxylation product in a bound form into an unbound form;
(7) adding 800 mu L acetonitrile (containing 1% formic acid) to terminate the enzymolysis reaction;
(8) centrifuging for 10 minutes at 12,000 × g using a high-speed refrigerated centrifuge;
(9) taking 400 mu L of centrifuged supernatant, and carrying out vacuum centrifugation and drying at 40 ℃;
(10) adding 60 μ L acetonitrile and 60 μ L water into the volatilized residue, and shaking to dissolve;
(11) the UPLC-MS/MS is adopted to detect the generation amount of 19-hydroxydeoxycholic acid, and the result shows that the 19-hydroxylation reaction of deoxycholic taurine is used as a probe, the generation rates of 19-hydroxylation products in liver microsomes of adult individuals with different sources are obviously different (figure 11), and the difference between the highest activity and the lowest activity is 270.6 times.
Claims (2)
1. A method for measuring cytochrome CYP3a7 enzyme activity for non-disease diagnostic purposes, comprising:
adding deoxycholic acid glycine as a reaction substrate into an incubation system with a cofactor NADPH or a generation system thereof, and detecting the generation amount of 19-hydroxydeoxycholic acid glycine in unit time to reflect the generation rate of a product; or adding deoxycholic acid taurine as a reaction substrate, and detecting the generation amount of 19-hydroxydeoxycholic acid taurine in unit time to reflect the generation rate of the product;
the structural formula of the deoxycholic glycine is shown as a formula (1):
the structural formula of the 19-hydroxydeoxycholic acid glycine is shown as a formula (2):
the structural formula of the deoxycholic taurine is shown as a formula (3):
the structural formula of the 19-hydroxydeoxycholic taurine is shown as a formula (4):
2. the method according to claim 1, wherein the method satisfies the following conditions:
(1) the concentration range of the substrate is 1-500 mu M;
(2) in an incubation system with pH of 7.4 and a cofactor NADPH or a generation system thereof, the reaction temperature is between 10 and 60 ℃;
(3) the reaction time is 5-120min, and the reaction is stopped when the generated 19-hydroxydeoxycholic glycine or 19-hydroxydeoxycholic taurine reaches the detection quantitative limit and the substrate conversion rate is lower than 20%.
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