Enzyme activity detection method of alcohol content related enzyme, enzyme activity-alcohol content model construction method and alcohol content prediction method

Abstract

The invention relates to the technical field of enzyme detection, in particular to a method for detecting enzyme activity in saliva, a method for constructing an enzyme activity-alcohol amount model and a method for predicting alcohol amount. The enzyme activity detection method comprises the following steps: S11, preparing an ethanol dehydrogenase reaction system and an acetaldehyde dehydrogenase reaction system; s12, obtaining an ethanol dehydrogenase determination group and an acetaldehyde dehydrogenase determination group; s13, obtaining an ethanol dehydrogenase blank group and an acetaldehyde dehydrogenase blank group; s14, reacting different determination groups and blank groups at the temperature of 20-40 DEG C for 1 minute; and S15, determining the absorbance of different determination groups and the corresponding blank groups at 340 nm by using a microplate reader, and calculating the enzyme activity according to the increased absorbance of the determination groups relative to the corresponding blank groups. The enzyme activity detection method disclosed by the invention is short in required time, simple to operate and suitable for large-flux sample detection, and a solution is easy to prepare.

Description

Technical Field

[0001] The present application relates to the technical field of enzyme detection, and in particular to a method for detecting the activity of alcohol-related enzymes, a method for constructing an enzyme activity-alcohol tolerance model, and a method for predicting alcohol tolerance. Background Art

[0002] The main component of alcohol is ethanol. The liver is the primary site of ethanol metabolism, with over 90% of ethanol being oxidized in the liver and 2-10% being excreted through the kidneys and lungs. Ethanol is first oxidized to acetaldehyde in the liver, primarily through the involvement of three enzymes: (1) alcohol dehydrogenase: a cytosolic component that metabolizes ethanol when drinking small amounts of alcohol; (2) microsomal ethanol oxidation system (cytochrome P450): located in the smooth endoplasmic reticulum and dependent on this pathway when alcohol levels in the body are high; and (3) catalase: located in the peroxisomes and mitochondria of hepatocytes, but its contribution to ethanol metabolism is not significant.

[0003] Compared with blood, urine, etc., saliva has gradually become the focus of biomarker research due to its ease of acquisition, and has good diagnostic value in oral diseases, hepatitis, diabetes, kidney disease, neurodegenerative diseases and immunodeficiency diseases. Oral proteomics studies have revealed that at least 3,000 protein components can be identified in oral saliva. Alcohol dehydrogenase (ADH) can oxidize ethanol to acetaldehyde, while acetaldehyde dehydrogenase can further oxidize acetaldehyde to acetic acid, and then hydrolyze it into carbon dioxide and water. In addition to oral flora, the activity of human salivary alcohol dehydrogenase also comes from the oral mucosa and salivary glands. Human salivary acetaldehyde dehydrogenase (HsALDH) uses NAD + or NADP + As a cofactor in catalytic oxidation reactions, it can catalyze the oxidation of aromatic and long-chain aliphatic aldehydes to their corresponding acids, and it can also catalyze the hydrolysis of ester bonds. Because alcohol dehydrogenase and acetaldehyde dehydrogenase play important roles in the human body, the enzyme activities of alcohol dehydrogenase and acetaldehyde dehydrogenase in saliva are also of great significance as biomarkers.

[0004] Currently, there are few studies on the detection of alcohol dehydrogenase and acetaldehyde dehydrogenase activities in saliva. The activity of salivary alcohol dehydrogenase is determined spectrophotometrically by measuring the reduction of the substrate p-nitrosodimethylaniline (NDMA). Salivary acetaldehyde dehydrogenase activity is determined fluorometrically using the substrate 6-methoxy-2-naphthaldehyde, but this method is complex and time-consuming, making it unsuitable for high-throughput testing. Summary of the Invention

[0005] Based on this, the purpose of this application is to use a simple, fast and low-cost method to establish a method for detecting the enzyme activity of alcohol dehydrogenase and acetaldehyde dehydrogenase from saliva and apply it to predict "alcohol consumption". One or more embodiments of this application provide a method for detecting the enzyme activity of alcohol-related enzymes, a method for constructing an enzyme activity-alcohol consumption model and a method for predicting alcohol consumption.

[0006] The technical solution of this application includes the following:

[0007] A method for detecting the activity of an alcohol tolerance-related enzyme comprises the following steps:

[0008] S11, preparing an alcohol dehydrogenase reaction system and an acetaldehyde dehydrogenase reaction system;

[0009] The alcohol dehydrogenase reaction system includes: a buffer solution, NAD + solution and ethanol solution;

[0010] The acetaldehyde dehydrogenase reaction system comprises: the buffer solution, the NAD + solution, KCl solution and acetaldehyde solution;

[0011] S12, adding the saliva sample to be tested to the alcohol dehydrogenase reaction system and the acetaldehyde dehydrogenase reaction system, respectively, and mixing to obtain an alcohol dehydrogenase assay group and an acetaldehyde dehydrogenase assay group;

[0012] S13, adding water to the alcohol dehydrogenase reaction system and the acetaldehyde dehydrogenase reaction system and mixing them, respectively, to obtain an alcohol dehydrogenase blank group and an acetaldehyde dehydrogenase blank group;

[0013] S14, reacting the alcohol dehydrogenase assay group, the acetaldehyde dehydrogenase assay group, the alcohol dehydrogenase blank group, and the acetaldehyde dehydrogenase blank group at 20° C. to 40° C. for 1 min;

[0014] S15. Using a microplate reader, measure the absorbance of the different assay groups and their corresponding blank groups at 340 nm, and calculate the enzyme activity based on the increase in absorbance of the assay groups relative to the corresponding blank groups.

[0015] Furthermore, the calculation formula of the enzyme activity is as follows:

[0016] A= (1);

[0017] Wherein, A is the enzyme activity, in U / mL; X2 is the absorbance of the blank group; X1 is the absorbance of the assay group; and v is the volume of the saliva sample to be tested, in μL.

[0018] Furthermore, the volume of the saliva sample to be tested is 5 μL to 10 μL.

[0019] Furthermore, in the alcohol dehydrogenase reaction system, the buffer solution, the NAD + The volume ratio of the solution and the ethanol solution is (100-110): (15-25): (15-25);

[0020] In the acetaldehyde dehydrogenase reaction system, the buffer solution, the NAD + The volume ratio of the solution, the KCl solution and the acetaldehyde solution is (100-110): (15-25): (15-25): (1-10).

[0021] Furthermore, in the alcohol dehydrogenase reaction system, the NAD + The mass concentration of the solution is 2% to 2.5%, and the volume concentration of the ethanol solution is 10% to 15%; and / or,

[0022] In the acetaldehyde dehydrogenase reaction system, the NAD + The mass concentration of the solution is 1% to 1.5%, and the volume concentration of the acetaldehyde solution is 1% to 1.5%.

[0023] Furthermore, the molar concentration of the KCl solution is 0.6M~0.8M.

[0024] Furthermore, in the alcohol dehydrogenase reaction system and the acetaldehyde dehydrogenase reaction system, the pH of the buffer solution is 8.0-8.5; and / or,

[0025] The molar concentration of the buffer solution is 40 mM to 60 mM.

[0026] A method for constructing an enzyme activity-alcohol tolerance model comprises the following steps:

[0027] S21. Count the alcohol consumption of the subjects and divide them into three groups according to the following criteria: the low metabolizer group, whose daily single-time pure alcohol consumption is M1, M1≤40g; the medium metabolizer group, whose regular pure alcohol consumption is M2, 40<M2<100g; the high metabolizer group, whose regular pure alcohol consumption is M3, M3≥100g;

[0028] S22. Obtain a saliva sample from the subject and measure the activity of alcohol tolerance-related enzymes using the enzyme activity detection method described above; the alcohol tolerance-related enzymes include the following two: an alcohol dehydrogenase reaction system and an acetaldehyde dehydrogenase;

[0029] S23. Based on the statistical results of step S21 and the measurement results of step S22, a t-test is used to perform a significance analysis on different populations. The enzyme activities of the two alcohol tolerance-related enzymes in the three groups of people and their saliva samples are combined to obtain nine different levels of alcohol tolerance, and an enzyme activity-alcohol tolerance model is constructed.

[0030] Furthermore, the enzyme activity-alcohol consumption prediction model is:

[0031] When ADH ≥ 200 U / mL and ALDH ≥ 180 U / mL, alcohol tolerance is level nine;

[0032] ADH is 125-200U / mL, ALDH ≥180U / mL, and alcohol tolerance is level eight;

[0033] ADH≤125U / mL, ALDH≥180U / mL, alcohol tolerance is level seven;

[0034] ADH is 125-200 U / mL, ALDH is 120-180 U / mL, and alcohol tolerance is level six;

[0035] ADH ≥ 200 U / mL, ALDH 120-180 U / mL, alcohol tolerance is level five;

[0036] ADH≤125U / mL, ALDH 120-180U / mL, alcohol tolerance is level 4;

[0037] ADH≤125U / mL, ALDH≤120U / mL, alcohol tolerance is level three;

[0038] ADH is 125-200U / mL, ALDH ≤ 120U / mL, and alcohol tolerance is level 2;

[0039] ADH ≥ 200 U / mL, ALDH ≤ 120 U / mL, alcohol tolerance is level one;

[0040] Wherein, ADH refers to the enzymatic activity of the alcohol dehydrogenase, and ALDH refers to the enzymatic activity of the acetaldehyde dehydrogenase.

[0041] A method for predicting alcohol consumption comprises the following steps:

[0042] The enzyme activity detection method described above is used to obtain the enzyme activities of alcohol dehydrogenase and acetaldehyde dehydrogenase in the saliva of the subjects. The enzyme activity-alcohol tolerance model obtained by the above construction method is used for analysis to obtain the alcohol tolerance level of the subjects.

[0043] The technical solution of this application includes at least the following advantages:

[0044] 1) This application is the first to use ethanol and acetaldehyde as substrates for the determination of alcohol dehydrogenase and acetaldehyde dehydrogenase activity in saliva. Compared with the traditional method of using GC-MS to detect saliva exhaled gas, the procedure is simpler and faster;

[0045] 2) The method for rapid detection of enzyme activity provided in this application is time-saving, does not require the addition of mercaptoethanol and DTT to the saliva sample to maintain its stability, can be performed at room temperature, does not require temperature control, is low-cost, simple to operate, and has a rapid reaction. The reagents required for the assay are easy to prepare, making it suitable for the detection of large-throughput samples.

[0046] 3) The alcohol dehydrogenase and acetaldehyde dehydrogenase activities measured in this application can be used to evaluate the sampler's ability to metabolize alcohol and further predict the drinking ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0048] Figure 1 Figure 2 shows the relationship between alcohol dehydrogenase (ADH) and alcohol metabolism capacity. Alcohol metabolism capacity is categorized as low, moderate, and high. Low metabolizers consume ≤ 40 g of pure alcohol per day, moderate metabolizers consume 40-100 g of pure alcohol per day, and high metabolizers consume ≥ 100 g of pure alcohol per day (pure alcohol / g = alcohol consumption / mL × alcohol content / %Vol. × 0.8). ADH activity of ≤ 125 U / mL indicates low alcohol metabolism capacity, 125-200 U / mL indicates moderate alcohol metabolism capacity, and ≥ 200 U / mL indicates high alcohol metabolism capacity. * indicates a significant difference (P < 0.05), ** indicates a highly significant difference (P < 0.01), and **** indicates an extremely significant difference (P < 0.0001).

[0049] Figure 2Figure 2 shows the relationship between aldehyde dehydrogenase (ALDH) and alcohol metabolism capacity. Alcohol metabolism capacity is categorized as low, medium, and high. Low metabolizers consume ≤ 40 g of pure alcohol per day, medium metabolizers consume 40-100 g of pure alcohol per day, and high metabolizers consume ≥ 100 g of pure alcohol per day (pure alcohol / g = alcohol consumption / mL × liquor alcohol content / %Vol. × 0.8). ADH activity of ≤120 U / mL indicates low alcohol metabolism capacity, 120-180 U / mL indicates medium alcohol metabolism capacity, and ≥180 U / mL indicates high alcohol metabolism capacity. * indicates a significant difference (P < 0.05), and **** indicates an extremely significant difference (P < 0.0001). DETAILED DESCRIPTION

[0050] Below in conjunction with embodiment and example, further elaborate the application.It should be understood that these examples are only used to illustrate the application and are not used to limit the scope of the application.In addition, it should be understood that after reading the content taught in this application, those skilled in the art can make various changes or modifications to the application, and these equivalent forms also fall within the protection scope of the claims appended hereto.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0052] the term

[0053] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0054] The term "and / or" as used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, wherein the any and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and technical solutions connected by "logical or".

[0055] In this application, "further" is used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0056] In this application, the terms "first," "second," "third," and "fourth," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, the terms "first," "second," "third," and "fourth," etc., are used only for non-exhaustive enumeration and description purposes and should not constitute closed-ended limitations on quantity.

[0057] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0058] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution within the numerical interval is considered continuous and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as every numerical value between these two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two numerical endpoints of the numerical range, as well as every integer between the two endpoints. In addition, when multiple ranges are provided to describe a feature or characteristic, these ranges can be combined. In other words, unless otherwise specified, ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0059] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that constant temperature treatment allows for temperature fluctuations within the precision range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0060] In this application, weight can be mass units known in the biological field such as μg, mg, g, kg, etc.

[0061] In this application, molar concentration M refers to moles per liter; molar concentration mM refers to millimoles per liter.

[0062] The technical solution of the embodiment of the present application provides a method for detecting the activity of alcohol-related enzymes, comprising the following steps:

[0063] S11, preparing an alcohol dehydrogenase reaction system and an acetaldehyde dehydrogenase reaction system;

[0064] The alcohol dehydrogenase reaction system includes: buffer solution, NAD + solution and ethanol solution;

[0065] The acetaldehyde dehydrogenase reaction system includes: buffer solution, NAD + solution, KCl solution and acetaldehyde solution;

[0066] S12, adding the saliva sample to be tested to the alcohol dehydrogenase reaction system and the acetaldehyde dehydrogenase reaction system, respectively, and mixing to obtain an alcohol dehydrogenase assay group and an acetaldehyde dehydrogenase assay group;

[0067] S13, adding water to the alcohol dehydrogenase reaction system and the acetaldehyde dehydrogenase reaction system and mixing them, respectively, to obtain an alcohol dehydrogenase blank group and an acetaldehyde dehydrogenase blank group;

[0068] S14, reacting the alcohol dehydrogenase assay group, acetaldehyde dehydrogenase assay group, alcohol dehydrogenase blank group, and acetaldehyde dehydrogenase blank group at 20°C-40°C for 1 min;

[0069] S15. Using a microplate reader, measure the absorbance of the different assay groups and their corresponding blank groups in step S14 at 340 nm, and calculate the enzyme activity based on the increase in absorbance of the assay group relative to the corresponding blank group.

[0070] Furthermore, the calculation formula of enzyme activity is as follows:

[0071] A= (1);

[0072] Where A is the enzyme activity, unit is U / mL; X2 is the absorbance of the blank group; X1 is the absorbance of the assay group; v is the volume of the saliva sample to be tested, unit is μL.

[0073] Furthermore, the volume of the saliva sample to be tested is 5 μL~10 μL.

[0074] Furthermore, in the alcohol dehydrogenase reaction system, the buffer solution, NAD + The volume ratio of solution and ethanol solution is (100~110):(15~25):(5~10);

[0075] In the acetaldehyde dehydrogenase reaction system, buffer solution, NAD + The volume ratio of solution, KCl solution and acetaldehyde solution is (100~110):(15~25):(15~25):(1~10).

[0076] Furthermore, in the alcohol dehydrogenase reaction system, NAD + The mass concentration of the solution is 2% to 2.5%, and the volume concentration of the ethanol solution is 10% to 15%; and / or,

[0077] In the acetaldehyde dehydrogenase reaction system, NAD + The mass concentration of the solution is 1%~1.5%, and the volume concentration of the acetaldehyde solution is 1%~1.5%.

[0078] Furthermore, the molar concentration of the KCl solution is 0.6M~0.8M.

[0079] Furthermore, in the alcohol dehydrogenase reaction system and the acetaldehyde dehydrogenase reaction system, the pH of the buffer solution is 8.0-8.5; and / or,

[0080] The molar concentration of the buffer solution is 40mM~60mM.

[0081] The technical solution of the embodiment of the present application also provides a method for constructing an enzyme activity-alcohol tolerance model, comprising the following steps:

[0082] S21. Count the alcohol consumption of the subjects and divide them into three groups according to the following criteria: the low metabolizer group regularly drinks pure alcohol in an amount of M1, M1 ≤ 40g; the medium metabolizer group regularly drinks pure alcohol in an amount of M2, 40 < M2 < 100g; the high metabolizer group regularly drinks pure alcohol in an amount of M3, M3 ≥ 100g;

[0083] S22. Obtain a saliva sample from the subject and measure the activity of an alcohol tolerance-related enzyme using the enzyme activity detection method according to any one of claims 1 to 7; the alcohol tolerance-related enzymes include the following two: an alcohol dehydrogenase reaction system and an acetaldehyde dehydrogenase;

[0084] S23. Based on the statistical results of step S21 and the measurement results of step S22, a t-test is used to perform a significance analysis on different populations. The enzyme activities of the two alcohol tolerance-related enzymes in the three groups of people and their saliva samples are combined to obtain nine different levels of alcohol tolerance, and an enzyme activity-alcohol tolerance model is constructed.

[0085] In step S23, a t-test is used to perform a significance analysis between the different populations. By analyzing the differences in ADH and ALDH enzyme activities, the enzyme activity thresholds that significantly affect metabolic capacity are screened out and used as the basis for classification. For example, if the ADH enzyme activity of high metabolizers is generally ≥200 U / mL, while that of low metabolizers is ≤125 U / mL, these two values ​​may be selected as the dividing points.

[0086] Furthermore, the enzyme activity-alcohol consumption prediction model is:

[0087] When ADH ≥ 200 U / mL and ALDH ≥ 180 U / mL, alcohol tolerance is level nine;

[0088] ADH is 125-200U / mL, ALDH ≥180U / mL, and alcohol tolerance is level eight;

[0089] ADH≤125U / mL, ALDH≥180U / mL, alcohol tolerance is level seven;

[0090] ADH is 125-200 U / mL, ALDH is 120-180 U / mL, and alcohol tolerance is level six;

[0091] ADH ≥ 200 U / mL, ALDH 120-180 U / mL, alcohol tolerance is level five;

[0092] ADH≤125U / mL, ALDH 120-180U / mL, alcohol tolerance is level 4;

[0093] ADH≤125U / mL, ALDH≤120U / mL, alcohol tolerance is level three;

[0094] ADH is 125-200U / mL, ALDH ≤ 120U / mL, and alcohol tolerance is level 2;

[0095] ADH ≥ 200 U / mL, ALDH ≤ 120 U / mL, alcohol tolerance is level one;

[0096] ADH refers to the enzymatic activity of alcohol dehydrogenase, and ALDH refers to the enzymatic activity of acetaldehyde dehydrogenase.

[0097] Since high alcohol dehydrogenase activity and low acetaldehyde dehydrogenase activity will cause acetaldehyde to be produced quickly but not metabolized quickly, the alcohol tolerance rating obtained by combining alcohol dehydrogenase and acetaldehyde dehydrogenase can achieve the purpose of predicting drinking ability.

[0098] The technical solution of the embodiment of the present application also provides a method for predicting alcohol consumption, comprising the following steps:

[0099] The enzyme activity detection method described above is used to obtain the enzyme activities of alcohol dehydrogenase and acetaldehyde dehydrogenase in the saliva of the subjects. The enzyme activity-alcohol tolerance model obtained by the above construction method is used for analysis to obtain the alcohol tolerance level of the subjects.

[0100] In some embodiments, the prediction of the subject's alcohol consumption can be made by referring to the levels and symptoms in Table 1.

[0101] Table 1 Correspondence between drinking capacity levels and symptoms

[0102]

[0103] The following are some specific examples.

[0104] For experimental parameters not specified in the following specific examples, reference is made to the guidance given in this application document, and reference may also be made to experimental manuals in the art or other experimental methods known in the art, or to the experimental conditions recommended by the manufacturer.

[0105] The raw materials and reagents involved in the following specific examples can be obtained commercially, or can be prepared by those skilled in the art according to known methods.

[0106] Example 1

[0107] 1. Sample collection and preparation of enzyme activity reagents

[0108] The method for collecting saliva samples from subjects is as follows:

[0109] Have the subject rinse their mouth with water 15 minutes before saliva collection. During saliva collection, slowly spit the required amount of saliva into the funnel. Tighten the cap of the sampling tube, mark it, and store it on ice until ready for use within 6 hours.

[0110] The reagent preparation method is as follows:

[0111] 50 mM Tris-HCl 8.0 buffer: Weigh 6.05 g Tris, dissolve it in water, adjust the pH to 8.0 with HCl, and make up the final volume to 1 L.

[0112] 1% NAD + Solution: Weigh 0.4 g NAD and dissolve it in water to make up to 40 mL;

[0113] 1% acetaldehyde solution: dilute 40% acetaldehyde solution to 1% with ultrapure water;

[0114] 0.75 M KCl solution: Weigh 18 g of KCl and dissolve it in water to make up to 500 mL.

[0115] 2% NAD + Solution: Weigh 0.4 g NAD and dissolve it in water to make up to 20 mL;

[0116] 10% ethanol solution: Dilute anhydrous ethanol to 10% with ultrapure water for later use.

[0117] 2. Alcohol dehydrogenase activity assay in 285 people

[0118] Step 1: Saliva samples were collected from 285 individuals and placed on ice for measurement within 6 hours. The samplers were asked to fill out a questionnaire about their drinking habits. The subjects were divided into three groups according to the following criteria: the single daily pure alcohol volume for the low metabolizer group was M1, M1≤40g; the single daily pure alcohol volume for the medium metabolizer group was M2, 40g<M2<100g; the single daily pure alcohol volume for the high metabolizer group was M3, M3≥100g.

[0119] Step 2: Prepare the following alcohol dehydrogenase reaction system: 10.5 mL Tris-HCl (pH 8.0), 2 mL 2% NAD + Solution, 2 mL 10% ethanol solution. The reaction solution system for a single sample is as follows: 105 μL Tris-HCl (pH 8.0), 20 μL 2% NAD +solution, 20 μL of 10% ethanol solution.

[0120] Step 3: Add 5 μL of saliva samples from 285 individuals to a 96-well plate. Set up three replicates for each sample. Use a dispenser to add 145 μL of the reaction solution from Step 2 to each well. Set up a blank control group with water instead of saliva.

[0121] Step 4: Incubate the mixed system at room temperature for 1 minute to allow the enzyme to react with the substrate. Measure the absorbance of the system at 340 nm using a microplate reader. Record the absorbance of the blank group as X1, and the absorbance of the test group as X2. Determine the alcohol dehydrogenase activity as the increase in enzyme activity units in the test group relative to the blank group within 1 minute.

[0122] Enzyme activity calculation: A= .

[0123] A represents the enzyme activity of saliva, the unit is U / mL, X1 is the absorbance of the blank group, X2 is the absorbance of the test group, and based on the input amount, the v value is 5.

[0124] 3. Construction of a model for the relationship between alcohol dehydrogenase activity and alcohol metabolism capacity

[0125] The results of the alcohol dehydrogenase test were compared with the alcohol consumption in the questionnaire, and the t-test was used to conduct a significant analysis between different groups. Figure 1 The results show that among 285 individuals, alcohol metabolism was categorized into three groups: low, medium, and high metabolizers. Alcohol metabolism capacity was positively correlated with alcohol dehydrogenase activity. Low metabolizers typically consumed ≤40 g of pure alcohol, medium metabolizers 40-100 g, and high metabolizers ≥100 g (pure alcohol / g = alcohol consumption / mL × liquor alcohol content / %Vol. × 0.8). Alcohol dehydrogenase activity of ≤125 U / mL was considered low alcohol metabolism, 125-200 U / mL was considered medium alcohol metabolism, and ≥200 U / mL was considered high alcohol metabolism. * indicates a significant difference (P < 0.05), ** indicates a highly significant difference (P < 0.01), and **** indicates an extremely significant difference (P < 0.0001).

[0126] 4. Determination of acetaldehyde dehydrogenase activity in 285 people

[0127] Step 1: Same as step 1 in section 2.285 “Determination of human alcohol dehydrogenase activity”.

[0128] Step 2: Prepare the following aldehyde dehydrogenase reaction system: 10 mL Tris-HCl (pH 8.0), 2 mL 0.75 MKCl solution, 2 mL 1% NAD + The reaction mixture for a single sample is as follows: 100 μL Tris-HCl (pH 8.0), 20 μL 0.75 M KCl solution, 20 μL 1% NADPH. + solution, 5 μL 1% acetaldehyde solution.

[0129] Step 3: Determine salivary aldehyde dehydrogenase activity: Add 5 μL of saliva samples from 285 individuals to a 96-well plate. Set up three replicates for each sample. Use a dispenser to add 145 μL of the reaction solution from Step 2 to each well. Set up a blank control with water instead of saliva.

[0130] Step 4: Incubate the mixed system at room temperature for 1 minute to allow the enzyme to react with the substrate. Measure the absorbance of the system at 340 nm using a microplate reader. Record the absorbance of the blank group as X1, and the absorbance of the test group as X2. Determine the aldehyde dehydrogenase activity as the increase in enzyme activity units in the test group relative to the blank group within 1 minute.

[0131] Enzyme activity calculation: A= .

[0132] A represents the enzyme activity of saliva, the unit is U / mL, X1 is the absorbance of the blank group, X2 is the absorbance of the test group, and based on the input amount, the v value is 5.

[0133] 5. Construction of a model for the relationship between acetaldehyde dehydrogenase activity and alcohol metabolism

[0134] The results of aldehyde dehydrogenase were compared with the alcohol consumption in the questionnaire, and the t-test was used to conduct a significant analysis between different groups. Figure 2 The results show that among 285 individuals, alcohol metabolism was categorized into three groups: low, intermediate, and high metabolizers. Alcohol metabolism was positively correlated with alcohol dehydrogenase activity. Low metabolizers typically consumed ≤40 g of pure alcohol, intermediate metabolizers 40-100 g, and high metabolizers ≥100 g (pure alcohol / g = alcohol consumption / mL × alcohol content / %Vol. × 0.8). Aldehyde dehydrogenase activity of ≤120 U / mL was considered low alcohol metabolism, 120-180 U / mL was considered medium alcohol metabolism, and ≥180 U / mL was considered high alcohol metabolism. * indicates a significant difference (P < 0.05), and **** indicates an extremely significant difference (P < 0.0001).

[0135] 6. Constructing an enzyme activity-alcohol tolerance model

[0136] After alcohol is ingested, it's metabolized by the enzymes alcohol dehydrogenase and acetaldehyde dehydrogenase. High alcohol dehydrogenase activity indicates strong ethanol metabolism, making intoxication less likely. High acetaldehyde dehydrogenase activity also indicates strong acetaldehyde metabolism, preventing acetaldehyde accumulation. Acetaldehyde is a carcinogen, and its harmful effects on the body are more severe than those on ethanol. Therefore, acetaldehyde dehydrogenase activity is used as the primary factor in evaluating drinking capacity.

[0137] Acetaldehyde dehydrogenase activity ≥180 U / mL indicates high acetaldehyde metabolism. As ethanol metabolism decreases, so does alcohol consumption. People in these three levels are less likely to accumulate acetaldehyde. Lower alcohol dehydrogenase activity increases the risk of intoxication, but slowly consuming alcoholic beverages poses minimal risk of harm.

[0138] An ALDH activity of 120-180 U / mL indicates moderate acetaldehyde metabolism. If the ADH activity is 125-200 U / mL, this person metabolizes ethanol and acetaldehyde at a relatively fast rate. If the ADH activity is ≥200 U / mL, this person experiences minimal acetaldehyde accumulation and is less susceptible to intoxication. If the ADH activity is ≤120 U / mL, rapid alcohol consumption can lead to alcohol poisoning.

[0139] Acetaldehyde dehydrogenase activity ≤120 U / mL: Low acetaldehyde metabolism. If alcohol dehydrogenase activity is ≤120 U / mL, this person's ethanol and acetaldehyde metabolism abilities are similar, and the rates are both very low, so drinking slowly has little impact on the body. If alcohol dehydrogenase activity is 125-200 U / mL, acetaldehyde accumulates slightly, and drinking slowly is unlikely to cause intoxication. If alcohol dehydrogenase activity is ≥200 U / mL, this person metabolizes ethanol very quickly, resulting in a large accumulation of acetaldehyde, which is extremely harmful to the body.

[0140] Example 2

[0141] Since the test subjects have never drunk alcohol, in order to assess their drinking ability, the alcohol consumption prediction method of Example 1 of the present application is used to predict their alcohol consumption: the salivary alcohol dehydrogenase and acetaldehyde dehydrogenase enzyme activities are tested according to the detection method described in Example 1, and the alcohol consumption is evaluated according to the alcohol consumption prediction model proposed in Example 1.

[0142] The test results showed that the candidate's alcohol dehydrogenase activity was 145.5 U / mL and acetaldehyde dehydrogenase activity was 173.0 U / mL. This indicates that the candidate has moderate ethanol and acetaldehyde metabolism abilities, resulting in a drinking capacity of Level 6. This type of person has similar ethanol and acetaldehyde metabolism abilities, and the metabolism rate is relatively fast. Drinking small amounts of alcohol can easily lead to intoxication, and acetaldehyde can be metabolized simultaneously. Therefore, it is recommended that the candidate drink small amounts of alcohol per day and consume alcohol slowly.

[0143] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0144] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0145] The above embodiments only express several implementation methods of the present application, but they should not be understood as limiting the scope of the patent application. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent in this application should be based on the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for detecting the activity of an alcohol tolerance-related enzyme, characterized in that: The steps include: S11, preparing an alcohol dehydrogenase reaction system and an acetaldehyde dehydrogenase reaction system; The alcohol dehydrogenase reaction system includes: a buffer solution, NAD + solution and ethanol solution; The acetaldehyde dehydrogenase reaction system comprises: the buffer solution, the NAD + solution, KCl solution and acetaldehyde solution; S12, adding the saliva sample to be tested to the alcohol dehydrogenase reaction system and the acetaldehyde dehydrogenase reaction system, respectively, and mixing to obtain an alcohol dehydrogenase assay group and an acetaldehyde dehydrogenase assay group; S13, adding water to the alcohol dehydrogenase reaction system and the acetaldehyde dehydrogenase reaction system and mixing them, respectively, to obtain an alcohol dehydrogenase blank group and an acetaldehyde dehydrogenase blank group; S14, reacting the alcohol dehydrogenase assay group, the acetaldehyde dehydrogenase assay group, the alcohol dehydrogenase blank group, and the acetaldehyde dehydrogenase blank group at 20° C. to 40° C. for 1 min; S15. Using a microplate reader, measure the absorbance of the different assay groups and their corresponding blank groups at 340 nm, and calculate the enzyme activity based on the increase in absorbance of the assay groups relative to the corresponding blank groups.

2. The enzyme activity detection method according to claim 1, characterized in that The calculation formula of the enzyme activity is as follows: A= （1）； Wherein, A is the enzyme activity, in U / mL; X2 is the absorbance of the blank group; X1 is the absorbance of the assay group; and v is the volume of the saliva sample to be tested, in μL.

3. The enzyme activity detection method according to claim 1, characterized in that The volume of the saliva sample to be tested is 5 μL to 10 μL.

4. The enzyme activity detection method according to any one of claims 1 to 3, characterized in that In the alcohol dehydrogenase reaction system, the buffer solution, the NAD + The volume ratio of the solution and the ethanol solution is (100-110): (15-25): (15-25); In the acetaldehyde dehydrogenase reaction system, the buffer solution, the NAD + The volume ratio of the solution, the KCl solution and the acetaldehyde solution is (100-110): (15-25): (15-25): (1-10).

5. The enzyme activity detection method according to claim 4, characterized in that In the alcohol dehydrogenase reaction system, the NAD + The mass concentration of the solution is 2% to 2.5%, and the volume concentration of the ethanol solution is 10% to 15%; and / or, In the acetaldehyde dehydrogenase reaction system, the NAD + The mass concentration of the solution is 1% to 1.5%, and the volume concentration of the acetaldehyde solution is 1% to 1.5%.

6. The method for detecting enzyme activity according to any one of claims 1 to 3 and 5, wherein: The molar concentration of the KCl solution is 0.6M~0.8M.

7. The method for detecting enzyme activity according to any one of claims 1 to 3 and 5, characterized in that: In the alcohol dehydrogenase reaction system and the acetaldehyde dehydrogenase reaction system, the pH of the buffer solution is 8.0-8.5; and / or, The molar concentration of the buffer solution is 40 mM to 60 mM.

8. A method for constructing an enzyme activity-alcohol tolerance model, characterized in that: The steps include: S21. Count the alcohol consumption of the subjects and divide them into three groups according to the following criteria: the low metabolizer group has a single daily pure alcohol consumption of M1, M1≤40g; the medium metabolizer group has a single daily pure alcohol consumption of M2, 40g<M2<100g; the high metabolizer group has a single daily pure alcohol consumption of M3, M3≥100g; S22. Obtain a saliva sample from the subject, and measure the activity of an alcohol tolerance-related enzyme using the enzyme activity detection method according to any one of claims 1 to 7; the alcohol tolerance-related enzymes include the following two: an alcohol dehydrogenase reaction system and an acetaldehyde dehydrogenase; S23. Based on the statistical results of step S21 and the measurement results of step S22, a t-test is used to perform a significance analysis on different populations. The enzyme activities of the two alcohol tolerance-related enzymes in the three groups of people and their saliva samples are combined to obtain nine different levels of alcohol tolerance, and an enzyme activity-alcohol tolerance model is constructed.

9. The construction method according to claim 8, characterized in that: The enzyme activity-alcohol consumption prediction model is: When ADH ≥ 200 U / mL and ALDH ≥ 180 U / mL, alcohol tolerance is level nine; ADH is 125-200U / mL, ALDH ≥180U / mL, and alcohol tolerance is level eight; ADH≤125U / mL, ALDH≥180U / mL, alcohol tolerance is level seven; ADH is 125-200 U / mL, ALDH is 120-180 U / mL, and alcohol tolerance is level six; ADH ≥ 200 U / mL, ALDH 120-180 U / mL, alcohol tolerance is level five; ADH≤125U / mL, ALDH 120-180U / mL, alcohol tolerance is level 4; ADH≤125U / mL, ALDH≤120U / mL, alcohol tolerance is level three; ADH is 125-200U / mL, ALDH ≤ 120U / mL, and alcohol tolerance is level 2; ADH ≥ 200 U / mL, ALDH ≤ 120 U / mL, alcohol tolerance is level one; Wherein, ADH refers to the enzymatic activity of the alcohol dehydrogenase, and ALDH refers to the enzymatic activity of the acetaldehyde dehydrogenase.

10. A method for predicting alcohol consumption, characterized in that: The steps include: The enzyme activity detection method according to any one of claims 1 to 7 is used to obtain the enzyme activity of alcohol dehydrogenase and acetaldehyde dehydrogenase in the saliva of the subject, and the enzyme activity-alcohol tolerance model obtained by the construction method according to claim 8 is used for analysis to obtain the alcohol tolerance level of the subject.

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