Method for reducing ethanol detection interference rate in animal experiment

Abstract

The invention discloses a method for reducing the interference rate of ethanol detection in animal experiments, which comprises the following steps: puncturing external jugular veins of an experimental rat, implanting a stress-free indwelling needle, feeding the experimental rat with white spirit, and collecting a blood sample according to a preset time point; drawing a standard curve according to the peak areas of ethanol and acetaldehyde and the concentration of the standard solution; and determining the peak areas of ethanol and acetaldehyde in the blood sample by adopting a headspace sampling mode, and calculating the concentrations of ethanol and acetaldehyde in the blood sample according to the established standard curve. The method can effectively solve the problem of stress reaction of experimental animals caused by multiple times of puncture blood sampling, so that the influence of the stress reaction on the accuracy of alcohol metabolism data is overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical detection, and particularly relates to a method for reducing the interference rate of ethanol detection in animal experiments, which is used to accurately monitor the change law of blood ethanol concentration after drinking alcohol. Background Art

[0002] The main component of wine is ethanol. Ethanol is mainly metabolized into acetaldehyde through the ethanol dehydrogenase (ADH) pathway in the liver in vivo, and then is converted into acetic acid by acetaldehyde dehydrogenase (ALDH). By measuring the concentrations of ethanol and acetaldehyde in blood, the alcohol metabolism ability of the same individual to different beverage wines can be directly reflected. The most common evaluation method in the current industry is: under the condition of administering an appropriate dose of wine to a model animal, using the headspace-gas chromatography test method to evaluate the contents of ethanol and acetaldehyde in blood at key time points within 24 hours after drinking, so as to obtain the post-drinking alcohol metabolism ability of the same individual for different beverage wines.

[0003] Currently, orbital or tail vein blood collection is mostly used in alcohol metabolism experiments. Multiple puncture blood collections will frequently stimulate animals, leading to stress responses in experimental animals and causing metabolic disorders, such as a surge in adrenaline, abnormal metabolic rates, such as elevated cortisol levels and abnormal liver enzyme activities, thus affecting the actual metabolic state of animals, interfering with the true reflection of blood ethanol concentration, and making it difficult to repeat the experimental results.

[0004] However, using the method of indwelling needles for blood collection also has its limitations. Because existing indwelling needles are mostly used in clinical humans and lack an adaptation design for small experimental animals, it is easy for the catheter to fall off or become blocked by blood coagulation due to animal activities. Some animal indwelling needle techniques, such as the jugular vein indwelling device, are mostly used for drug infusion or long-term physiological detection, but there are problems such as easy catheter detachment and anticoagulants interfering with ethanol detection. For example, the conventional anticoagulant sodium heparin may indirectly affect the ethanol metabolism rate by inhibiting enzyme activity, resulting in distorted data.

[0005] Therefore, there is an urgent need to develop a more reasonable blood collection and detection method that can be used to accurately monitor the change law of blood ethanol concentration after drinking alcohol. Summary of the Invention

[0006] The main purpose of the present invention is to provide a method for reducing the interference rate of ethanol detection in animal experiments, which can effectively solve the stress response of experimental animals caused by multiple puncture blood collections, thereby overcoming the influence of this stress response on the accuracy of alcohol metabolism data.

[0007] A method for reducing the interference rate of ethanol detection in animal experiments includes the following steps:

[0008] Step 1: Puncture the external jugular vein of the experimental rats, implant a stress-free indwelling needle. After feeding them with white liquor, collect blood samples at preset time points. The stress-free indwelling needle uses a biocompatible silicone catheter to connect to the jugular indwelling steel needle. The end of the catheter is integrated with a micro-unidirectional valve to prevent blood reflux and air embolism. The unidirectional valve and the elastic connecting tube are adapted to the activity requirements of animals of different body sizes. The end of the catheter is connected to a three-way valve, and an external liquid storage bag pre-filled with anticoagulant is connected.

[0009] Step 2: Draw a standard curve based on the peak areas of ethanol and acetaldehyde and the standard solution concentration. Record the peak area values of ethanol and acetaldehyde in the test sample and the spiked sample. Perform linear regression with the peak areas of ethanol and acetaldehyde in the spiked sample as the ordinate and the mass concentrations of ethanol and acetaldehyde in the spiked sample as the abscissa to obtain a linear equation.

[0010] Step 3: Dilute the blood samples collected in Step 1 to 0.5 mL with ddH2O, put them into headspace vials, and use the headspace injection method to measure the peak areas of ethanol and acetaldehyde. Calculate the concentrations of ethanol and acetaldehyde in the blood according to the standard curve established in Step 2.

[0011] Preferably, Step 1 includes: (1) Preoperative preparation: After fasting the experimental rats for 12 hours, anesthetize and fix them, shave the hair on the neck and back, and disinfect with iodophor. Catheter implantation: Puncture the external jugular vein of the experimental rats, implant the indwelling steel needle and connect the catheter. Lead the catheter through a subcutaneous tunnel to the back. Inject 0.1 ml of anticoagulant to pre-fill the catheter.

[0012] (2) Feeding with white liquor: Feed the white liquor 24 hours after implanting the indwelling steel needle. Each liquor sample corresponds to a group of animals, with at least 10 animals in each group. Quantitatively intragastrically administer the white liquor at a dose based on the standard of 0.14 mL / 10 g body weight.

[0013] (3) Blood collection: Collect blood samples at the preset time points of 0, 15, 30, 60, 120, 180, 240, 300, and 360 minutes after drinking. Immediately inject anticoagulant to flush the catheter after each blood collection to prevent thrombus formation.

[0014] Preferably, the anticoagulant includes 0.5% sodium citrate, 0.1% EDTA-K2, and 1% trehalose to avoid inhibiting the activity of alcohol dehydrogenase. The anticoagulant-to-blood volume ratio is 1:15 to reduce the influence of dilution effect on ethanol concentration.

[0015] Preferably, in Step 2, prepare a series of concentration ethanol and acetaldehyde standard solutions, and take one portion of the ethanol and acetaldehyde standard solutions equal to the sample volume for each concentration to obtain a series of concentration spiked samples.

[0016] Preferably, in step three, the conditions for headspace injection determination are as follows: the detector temperature is 250 °C, the injection port temperature is 240 °C; the constant flow mode is 8.0 mL / min; the split ratio is 1:1; the N2 flow rate is 8 mL / min, the H2 flow rate is 45 mL / min, and the air flow rate is 450 mL / min; the peak elution time is: acetaldehyde 1.40 min / L, ethanol 3.30 min / L.

[0017] Preferably, in step three, the initial temperature of the column temperature of the chromatographic column is 40 °C, which is raised to 60 °C at a rate of 10 °C / min, then raised to 120 °C at a rate of 20 °C / min, and then raised to 240 °C at a rate of 40 °C / min. The headspace temperature is 55 °C, and the heating time is 30 min.

[0018] Preferably, the stress-free indwelling needle further includes a fixing device, which comprises a water-absorbing pad and an X-shaped adjustable silicone band, and is fixed on the back of the experimental rat to ensure that the catheter fits the skin without displacement.

[0019] Preferably, the water-absorbing pad includes a benzalkonium chloride anti-inflammatory layer.

[0020] The present invention overcomes the use of an indwelling needle system with a specific design, solves the problem of stress response in experimental animals caused by multiple punctures for blood collection, and at the same time uses a specific anticoagulant to solve the potential interference effect between the existing anticoagulation scheme of the indwelling needle and the activity of alcohol-metabolizing enzymes, improving the accuracy of alcohol metabolism data detection. The method of the present invention can be used to screen effective hangover raw materials, which helps to guide the development of "low-intoxication" liquor products. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic structural diagram of the stress-free indwelling needle adopted by the present invention;

[0022] Among them: 1 - indwelling steel needle, 2 - indwelling catheter, 3 - micro check valve, 4 - extension tube, 5 - sealing clip, 6 - blood sampling port, 7 - sealing cap, 8 - anticoagulant storage sac. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of the present invention belong. If the definitions stated in this section are contrary to or otherwise inconsistent with the definitions stated in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions listed in this section shall prevail over the definitions incorporated herein by reference.

[0025] Unless otherwise specified, the methods used in the following embodiments are all conventional methods. The materials, reagents, and instruments used, unless otherwise specified, are all conventional materials, reagents, and instruments in this field, and those skilled in the art can obtain them through commercial channels. When an equivalent, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values and lower preferred values, it should be understood that all ranges formed by any pairing of any upper range limit or preferred value and any lower range limit or preferred value are specifically disclosed, regardless of whether the range is separately disclosed. For example, when the range "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its endpoint values and all integers and fractions within the range. In the specification and claims of this application, range limitations can be combined and / or interchanged, and if not otherwise stated, these ranges include all sub-ranges subsumed therein.

[0026] Reference Figure 1 As shown in the reference, the stress-free indwelling needle adopted in the following embodiments is designed as follows: A biocompatible silicone indwelling catheter 2 (inner diameter 0.3 - 0.5 mm) is connected to a jugular vein indwelling steel needle 1. The end of the indwelling catheter 2 is integrated with a micro one-way valve 3 to prevent blood reflux and air embolism. The fixing device includes a water-absorbing pad (containing a benzalkonium chloride anti-inflammatory layer) and an X-shaped adjustable silicone band (not shown in the figure), which is fixed on the back of the rat to ensure that the indwelling catheter 2 fits the rat skin without displacement. It is matched with an elastic extension tube 4 with adjustable length to adapt to the activity requirements of animals of different body types. A sealing clip 5 is also provided on the extension tube 4. The blood sampling port 6 at its end is sealed with a sealing cap 7, and is externally connected to an anticoagulant reservoir 8 (volume 1 mL) pre-filled with anticoagulant.

[0027] The anticoagulant reservoir 8 is filled with a special anticoagulant, which contains 0.5% sodium citrate + 0.1% EDTA-K2 + 1% trehalose. This special anticoagulant effectively avoids inhibiting the activity of alcohol dehydrogenase. The anticoagulant-to-blood volume ratio is 1:15, and this ratio can reduce the influence of dilution effect on ethanol concentration.

[0028] The non-stress indwelling needle supports multiple blood samplings without repeated punctures. Through a negative pressure micropump, a small amount of blood (<50 μL / time) is automatically collected into a pre-cooled EP tube.

[0029] A method for reducing the interference rate of ethanol detection in animal experiments provided by the present invention includes the following steps:

[0030] Step 1: Preoperative preparation: After the experimental rats are fasted for 12 hours, they are anesthetized and fixed, the hair on the neck and back is shaved, and disinfected with iodophor.

[0031] Step 2: Catheter implantation: Puncture the external jugular vein, implant the indwelling steel needle 1 and connect the indwelling catheter 2; the indwelling catheter 2 is led to the back through a subcutaneous tunnel and cross-fixed on the back of the animal by an X-shaped silica gel band; inject 0.1 ml of anticoagulant to pre-fill the indwelling catheter 2.

[0032] Step 3: Feeding white wine: After 24 hours of implanting the indwelling needle, feed white wine to the experimental rats; each wine sample corresponds to a group of experimental rats, and there are at least 10 rats in each group; quantitatively intragastrically administer white wine (dose 0.14 mL / 10 g body weight).

[0033] Step 4: Blood sample collection: Collect blood samples through the blood sampling port 6 at preset time points (such as 0 / 15 / 30 / 60 / 120 / 180 / 240 / 300 / 360 minutes) after drinking; immediately inject anticoagulant (10 μL) to flush the catheter after each blood sampling to prevent thrombus formation.

[0034] Step 5: Detection and analysis:

[0035] 1. Plot the standard curve

[0036] 1.1 Chromatographic reference conditions

[0037] 1.1.1 Instrument model: Aoutosystem XL gas chromatograph, TurboMatrix 40 automatic headspace sampler, Perkin Elmer Corporation, or equivalent.

[0038] 1.1.2 Chromatographic column: Agilent J&W-GC type 30 m × 0.53 mm × 1.00 m silanized fused silica column or equivalent chromatographic column.

[0039] 1.1.3 Temperature programming: The initial temperature of the column is 40 °C, heated to 60 °C at 10 °C / min, held for 0 min, then heated to 120 °C at 20 °C / min, held for 0 min, and then heated to 240 °C at 40 °C / min, the headspace is 55 °C, and the heating time is 30 min.

[0040] 1.1.4 Detector temperature: 250 °C.

[0041] 1.1.5 Injection port temperature: 240 °C.

[0042] 1.1.6 Constant flow mode: 8.0 mL / min.

[0043] 1.1.7 Injection volume: Headspace injection.

[0044] 1.1.8 Split ratio: 1:1.

[0045] 1.1.9 Carrier gas flow rate: N2, 8 mL / min; H2, 45 mL / min; Air, 450 mL / min.

[0046] 1.1.10 Elution time: Acetaldehyde 1.40 min / L, Ethanol 3.30 min / L.

[0047] Note: The above are reference conditions, which can be adjusted according to the actual situations such as different brands of instruments and different samples.

[0048] 1.2 Preparation of standard samples

[0049] Refer to GB / T 42430, and quantitative analysis is carried out by the calibration curve method. The mass concentrations of ethanol in the prepared standard solutions are 0.10 mg / mL, 0.20 mg / mL, 0.50 mg / mL, 0.80 mg / mL, 1.00 mg / mL, 2.00 mg / mL, and 3.00 mg / mL.

[0050] Prepare a series of concentration ethanol and acetaldehyde standard solutions. Take one portion of ethanol and acetaldehyde standard solutions equal in amount to the sample for each concentration to obtain a series of concentration spiked samples, and operate in parallel with the sample. The concentration of the target substance in the sample should be within the linear range of the calibration curve. The concentrations of the prepared mass concentration solutions do not need to be strictly consistent, and the correlation coefficient (r) should not be less than 0.997. If the concentration of the sample exceeds the linear range, the sample needs to be diluted with water or a new calibration curve needs to be made to make the concentration of the sample within the linear range of the calibration curve.

[0051] 1.3 Establishment of calibration curve

[0052] Draw a calibration curve based on the peak areas of ethanol and acetaldehyde and the standard solution concentrations, record the peak area values of ethanol and acetaldehyde in the test sample and the spiked sample, and perform linear regression with the peak areas of ethanol and acetaldehyde in the spiked sample as the ordinate and the mass concentrations of ethanol and acetaldehyde in the spiked sample as the abscissa to obtain a linear equation.

[0053] 2. Index detection

[0054] Dilute the blood sample to 0.5 mL with ddH2O, put it into a headspace injection vial, adopt the headspace injection method, measure the peak areas of ethanol and acetaldehyde according to the 1.1 chromatographic reference conditions, and calculate the concentrations of ethanol and acetaldehyde in the whole blood according to the calibration curve established in 1.3.

[0055] Example 1: Effects of Different Blood Sampling Methods on Ethanol Metabolism in Experimental Animals

[0056] Using the method of the present invention, traditional blood sampling methods, and traditional anticoagulants, alcohol metabolism studies were carried out on the same white liquor (total acid 1.1 g / L, total ester 2.4 g / L). One hour after drinking, blood samples were taken from the experimental group and two groups of rats using traditional methods, and stress indicators, ethanol concentration in the blood, and other indicators were measured respectively, and CV (the ratio of the standard deviation to the mean) was calculated. One hour after drinking, blood samples were taken from the experimental group, the traditional heparin anticoagulant group, and the blank group of rats, and the ethanol concentration in the blood was measured. It was found that compared with the traditional blood sampling method, the stress indicators of the experimental animals using the method of the present invention were significantly decreased, and the coefficient of variation of ethanol concentration was reduced; compared with the traditional heparin anticoagulant, the interference rate of ethanol detection was significantly reduced.

[0057] Table 1 Related Stress Effect Data

[0058]

[0059] Table 2 Data Stability (Coefficient of Variation of Blood Ethanol Concentration (CV)), n = 20

[0060]

[0061] Table 3 Results of Ethanol Detection Interference Rate, n = 10

[0062]

[0063] From the above test results, it can be seen that using the indwelling needle system of the present invention reduces data fluctuations (CV = 3.5%), and improves the experimental reproducibility.

[0064] Example 2: Comparison of Metabolism Rates of Different Aromatic Types of White Liquor

[0065] Using the method of the present invention to monitor the metabolic differences of light-aromatic type (total acid 0.8 g / L, total ester 1.8 g / L) and sauce-aromatic type (total acid 2.3 g / L, total ester 3.6 / L, tetramethylpyrazine 5.3 mg / L) white liquor in rats, the following differences were found:

[0066] 1. Difference in peak time: The ethanol concentration in the light-aromatic type group reached the peak at 60 minutes (162.7 mg / dL), and the peak time of the sauce-aromatic type group was the same but the peak concentration was lower (148.9 mg / dL).

[0067] 2. Metabolism rate: The ethanol clearance half-life (t1 / 2) of the light-aromatic type group was 85 minutes, and that of the sauce-aromatic type group was 92 minutes, presumably related to the inhibition of the activity of alcohol dehydrogenase (ADH) by pyrazine substances in the sauce-aromatic type.

[0068] 3. Influence of flavor components: In light aroma type liquor, ethyl acetate may accelerate metabolism by promoting the activity of ADH, while in strong aroma type liquor, pyrazine substances may delay metabolism.

[0069] Table 4 Ethanol concentration data (mg / dL) in the blood of two groups of rats

[0070]

[0071]

[0072] The data of Example 2 show that the metabolism of liquors of different aroma types is different. Using the above detection method, products with different formulas can be designed, and the method of the present invention can be used to guide the development of "low intoxication type" products.

[0073] Example 3: Evaluation of the efficacy of anti-alcoholism drugs

[0074] Drug intervention: Puerarin tablets (dose 50 mg / kg), and alcohol (2 g / kg) was given 30 minutes after gavage.

[0075] Experimental grouping: Control group (without drug), puerarin intervention group.

[0076] Blood sampling method: The same indwelling needle system was used to avoid stress interference.

[0077] Table 5 Ethanol concentration data (mg / dL) in the blood

[0078]

[0079] The following key conclusions can be drawn from the above experiments:

[0080] 1. Metabolism acceleration effect: The ethanol concentration in the puerarin group was significantly lower than that in the control group after 120 minutes (105.8 vs. 129.5 mg / dL), and the half-life was shortened from 85 minutes to 61 minutes (p < 0.05).

[0081] 2. Mechanism of action: Puerarin may accelerate ethanol clearance by activating the activities of ADH and aldehyde dehydrogenase (ALDH) and reducing oxidative stress.

[0082] Example 3 shows that puerarin significantly shortens the ethanol half-life, suggesting that puerarin can be added to liquor to increase the metabolism of liquor.

[0083] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for reducing the interference rate of ethanol detection in animal experiments, characterized in that, It includes the following steps: Step 1: Puncture the external jugular vein of the experimental rats, implant a stress-free indwelling needle. After feeding them with white wine, collect blood samples at preset time points. The stress-free indwelling needle uses a biocompatible silicone catheter to connect to the jugular indwelling steel needle. The end of the catheter integrates a micro one-way valve to prevent blood reflux and air embolism. The one-way valve and the elastic connecting tube are adapted to the activity requirements of animals of different body sizes. The end of the catheter is connected to a three-way valve, and an anticoagulant-precharged liquid storage sac is externally connected. Step 2: Draw a standard curve based on the peak areas of ethanol and acetaldehyde and the standard solution concentration. Record the peak area values of ethanol and acetaldehyde in the test sample and the spiked sample. Perform linear regression with the peak areas of ethanol and acetaldehyde in the spiked sample as the ordinate and the mass concentrations of ethanol and acetaldehyde in the spiked sample as the abscissa to obtain a linear equation. Step 3: Dilute the blood sample collected in Step 1 to 0.5 mL with ddH2O, put it into a headspace vial, and use the headspace injection method to measure the peak areas of ethanol and acetaldehyde. Calculate the concentrations of ethanol and acetaldehyde in the blood according to the standard curve established in Step 2.

2. The method according to claim 1, wherein Step 1 includes: (1) Preoperative preparation: After the experimental rats are fasted for 12 hours, they are anesthetized and fixed, the hair on the neck and back is shaved, and disinfected with iodophor. Catheter implantation: Puncture the external jugular vein of the experimental rats, implant an indwelling steel needle and connect the catheter. The catheter is led to the back through a subcutaneous tunnel. Inject 0.1 ml of anticoagulant to pre-fill the catheter. (2) Feeding white wine: Feed white wine 24 hours after implanting the indwelling steel needle. Each wine sample corresponds to a group of animals, with at least 10 animals in each group. Quantitatively gavage white wine, and the dose is calculated according to the standard of 0.14 mL / 10 g body weight. (3) Blood collection: Collect blood samples at preset time points of 0, 15, 30, 60, 120, 180, 240, 300, and 360 minutes after drinking. Immediately inject anticoagulant to flush the catheter after each blood collection.

3. The method according to claim 2, wherein The anticoagulant includes 0.5% sodium citrate, 0.1% EDTA-K2, and 1% trehalose, and the volume ratio of the anticoagulant to the blood is 1:

15.

4. The method according to claim 1, wherein In Step 2, prepare a series of concentration ethanol and acetaldehyde standard solutions. Take one portion of the ethanol and acetaldehyde standard solutions with the same amount as the sample for each concentration to obtain a series of concentration spiked samples.

5. The method according to claim 1, wherein In Step 3, the conditions for headspace injection determination are: the detector temperature is 250 °C, the injection port temperature is 240 °C; the constant flow mode is 8.0 mL / min; the split ratio is 1:1; the N2 flow rate is 8 mL / min, the H2 flow rate is 45 mL / min, and the air flow rate is 450 mL / min; the peak time is: acetaldehyde 1.40 min / L, ethanol 3.30 min / L.

6. The method according to claim 1, characterized in that, In Step 3, the initial temperature of the column temperature of the chromatographic column is 40 °C, it is raised to 60 °C at a rate of 10 °C / min, then raised to 120 °C at a rate of 20 °C / min, and then raised to 240 °C at a rate of 40 °C / min. The headspace temperature is 55 °C, and the heating time is 30 min.

7. The method according to claim 1, characterized in that, The stress-free indwelling needle also includes a fixing device, and the fixing device includes a water-absorbing pad and an X-shaped adjustable silicone band.

8. The method according to claim 7, wherein The water-absorbing pad includes a benzalkonium chloride anti-inflammatory layer.

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