CN111829851A - Pretreatment method for determination of 18 amino acids in brain tissue and plasma of C57 mouse - Google Patents
Pretreatment method for determination of 18 amino acids in brain tissue and plasma of C57 mouse Download PDFInfo
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Abstract
The invention discloses a pretreatment method for measuring 18 amino acids in brain tissues and blood plasma of a C57 mouse, belonging to the field of pretreatment of endogenous substances of biological samples. The method uses an ultraviolet spectrophotometer and a high performance liquid chromatograph to investigate the method for treating the brain tissue homogenate and the plasma by adopting a liquid-liquid extraction combined protein precipitation method, mainly inspects the influence of a sample pretreatment method on the protein removal amount, the total lipid removal amount and the 18 amino acid extraction amount in a sample, and further optimizes to obtain the optimal pretreatment method for measuring the 18 amino acids in the brain tissue and the plasma of the C57 mouse.
Description
Technical Field
The invention belongs to the field of pretreatment of endogenous substances of biological samples, and particularly relates to a pretreatment method for measuring 18 amino acids in brain tissues and blood plasma of a C57 mouse.
Background
The quantitative determination of various endogenous markers in different biological samples plays an increasingly important role in the study of disease pathogenesis. The change of the content of amino acid playing the role of neurotransmitter in brain tissue is a hotspot of the research of diseases such as depression, cerebral ischemia and the like, and the related biological samples are mainly animal brain tissue and blood plasma. Before the endogenous amino acid is quantitatively determined, a biological sample needs to be pretreated by adopting a special pretreatment method, so that endogenous interfering substances are removed, and the endogenous amino acid to be determined is maximally reserved. Brain tissue and plasma contain a large amount of proteins, lipids, small molecules, salts, etc., and studies have shown that lipids in brain tissue account for 1/2 on a dry weight basis. The invention mainly aims at a pretreatment method for measuring amino acids in brain tissues and blood plasma. Common sample pretreatments include Protein Precipitation (PPT), liquid-liquid extraction (LLE), or Solid Phase Extraction (SPE). Since amino acids are a substance with a relatively high polarity and have a low solubility in organic solvents, liquid-liquid extraction methods for extracting amino acids using organic solvents are not suitable. Solid phase extraction methods achieve selective extraction of specific analytes by using different solid phase extractants and elution conditions. Based on the prior art, the method for simultaneously extracting 18 amino acids by adopting a solid phase extraction method has certain difficulties. In the prior pretreatment for amino acid measurement in a biological sample, a protein precipitation method is mainly adopted, and the advantages and disadvantages of the pretreatment method are examined according to the removal rate of protein and the extraction rate of amino acid, but the influence of lipid and other substances on the measurement is not considered, and the lipid interferes with the measurement and pollutes instruments when the substances to be measured are measured. Therefore, based on the existing amino acid treatment method, the invention establishes an evaluation system of the pretreatment method for measuring 18 amino acids in brain tissues and blood plasma of C57 mice, and further optimizes the pretreatment method so as to accurately and efficiently measure the amino acid content in the next step.
Disclosure of Invention
The invention aims to provide a pretreatment method for measuring 18 amino acids in brain tissues and plasma of a C57 mouse, which has the advantages of reducing the content of protein and lipid in a sample to be analyzed and ensuring the high-efficiency extraction of the amino acids, thereby facilitating the next measurement of the content of endogenous amino acids in the brain tissues and plasma. The invention establishes an evaluation system of a pretreatment method for measuring 18 amino acids in brain tissues and blood plasma of a C57 mouse, and obtains an optimal pretreatment method by observing the removal effect of lipid, protein and other impurities, thereby achieving the purposes of reducing more interfering substances in a sample and obtaining better amino acid extraction rate.
The samples described herein are C57 mouse cortex and plasma, the 18 amino acids are: glutathione (Gsh), aspartic acid (Asp), glutamic acid (Glu), serine (Ser), glutamine (Gln), citrulline (Cit), glycine (Gly), arginine (Arg), taurine (Tau), tyrosine (Tyr), alanine (Ala), gamma-aminobutyric acid (GABA), tryptophan (Trp), methionine (Met), valine (Val), phenylalanine (Phe), isoleucine (Ile), leucine (Leu)
The specific technical scheme of the invention is as follows:
a pretreatment method for measuring 18 amino acids in brain tissues and plasma of a C57 mouse comprises the following steps:
the sample involved in the patent is C57 mouse brain homogenate and plasma.
Step one, preparing brain tissue homogenate and blood plasma
Precisely weighing the brain tissue of a C57 mouse, and performing the following steps of 1: 5-1: adding ultrapure water into the mixture in a proportion of 20 (the weight of the brain tissue: the ultrapure water is mg: mu L), and homogenizing the mixture for 30-90 s by using a handheld homogenizer to obtain the brain tissue homogenate. The sample obtained by blood sampling from the orbit is centrifuged to obtain the supernatant, and the plasma to be processed is obtained.
Step two, liquid-liquid extraction of the delipidated sample
Taking 100-300 mul of sample, adding 100-300 mul of methyl tert-butyl ether, whirling and shaking, centrifuging, and discarding the upper layer to remove the lipid in the sample.
Step three, treating the sample by a protein precipitation method
And (3) taking 100-300 mu L of the degreased sample obtained in the second step, adding 100-300 mu L of protein precipitator, and removing the protein in the sample by vortex oscillation, centrifugation and supernatant taking.
Step four, investigating the total protein and total lipid content of the sample
And taking 100-300 mu L of the sample treated in the second step and the third step, measuring the absorbance of the sample at 280nm by using an ultraviolet spectrophotometer, and inspecting the total protein content in the treated brain tissue sample. Taking 100 mu L-300mu.L of the treated sample was placed in a glass test tube, the solvent was evaporated, 200. mu.L of concentrated H was added2SO4(96%), vortex mixing, heating in 90 deg.C water bath for 20min, rapidly cooling to room temperature, adding 200 μ L of 1 mg. mL-1Vanillin solution (prepared by 17% phosphoric acid solution), reacting at normal temperature in dark place, and measuring absorbance at 500-550 nm wavelength.
Step five, switching a fluorescence detector by an HPLC diode array detector (PDA) to inspect the impurity removal amount and the amino acid extraction amount
The liquid phase conditions of HPLC comprise chromatographic column, mobile phase A and mobile phase B, wherein the chromatographic column is octadecyl silane bonded silica gel column, the mobile phase A is methanol, and the mobile phase B is 30 mmol.L-1And a sodium acetate buffer solution with pH 6.20. The amino acids in brain tissue and blood plasma samples were measured after derivatization with o-phthalaldehyde at a sample size of 20 μ L and a flow rate of 0.9 mL/min-1The column temperature was 30 ℃. The detection wavelength of the PDA detector is 230nm, the excitation wavelength of the fluorescence detector is 340nm, and the emission wavelength is 450 nm.
Step six, determining the pretreatment conditions of the sample
Selecting a pretreatment method through the result of the fifth and sixth optimization, and directly selecting 0.1-0.5 mol.L-1The perchloric acid solution of (a) removes lipids and proteins from the sample. In order to further optimize the sample pretreatment method, the consumption of perchloric acid is optimized. According to the following steps of 1: 3-1: 9 (sample: perchloric acid solution ═ muL:. mu.L) ratio of 0.1 to 0.5 mol. mu.L-1And optimizing the sample treated by the perchloric acid to obtain the optimal dosage through the investigation of the fifth step and the investigation of the sixth step.
The invention has the beneficial effects that: the invention establishes an evaluation system of a pretreatment method for measuring 18 amino acids in brain tissues and blood plasma of C57 mice, and mainly evaluates the removal effect of the pretreatment method on interfering substances of lipid, protein and other substances and the extraction effect on the 18 amino acids by using an ultraviolet spectrophotometer to measure absorbance and an HPLC method. The optimal extraction method obtained by evaluating and optimizing the pretreatment method provides necessary conditions for accurately and efficiently measuring the amino acid in the sample.
Drawings
FIG. 1 graph of protein content in samples treated with acetonitrile and perchloric acid
FIG. 2 Absorbance at 522nm of different derivatization reaction times of lipids with vanillin
FIG. 3 is a graph of the total lipid content in samples after MTBE treatment
FIG. 4 chromatograms of perchloric acid and MTBE + perchloric acid-treated samples at 230nm
FIG. 5 chromatograms of acetonitrile and MTBE + acetonitrile treated samples at 230nm
FIG. 6 chromatograms of perchloric acid and acetonitrile treated samples at 230nm
FIG. 7 protein content plot of samples with different amounts of perchloric acid
FIG. 8 is a graph showing the lipid content of samples with different amounts of perchloric acid
FIG. 9 Effect of different amounts of perchloric acid on amino acid extraction
Detailed Description
The invention is further illustrated with reference to the following figures and examples without thereby limiting the scope of the invention.
Case one: brain homogenate and plasma sample pretreatment
A200. mu.L sample was homogenized, 200. mu.L of methyl tert-butyl ether was added, vortexed for 30s, centrifuged at 15000g at 4 ℃ for 15min, and the supernatant was discarded to remove most of the lipid in the sample.
Collecting 200 μ L of the above lipid-removed sample, adding 400 μ L acetonitrile, vortexing and shaking for 30s, centrifuging at 4 deg.C for 15min at 15000g, and collecting supernatant to remove protein in the sample; 200. mu.L of the above delipidated sample was added to 400. mu.L of 0.4 mol. L-1The perchloric acid solution was centrifuged at 15000g at 4 ℃ for 15min for 30 seconds by vortexing, and the supernatant was removed to remove the protein in the sample.
Case two: evaluation of pretreatment method
Taking a certain amount of the treated sample, and measuring the absorbance of the sample at 280nm by using an ultraviolet spectrophotometer, wherein the absorbance is in direct proportion to the protein content. FIG. 1 illustrates: perchloric acid is more effective in removing proteins from the sample.
Placing 200 μ L of the treated sample in a glass test tube, evaporating solvent, adding 200 μ L of concentrated H2SO4(96%), vortexed for 30s, heated in a water bath at 90 ℃ for 20min, rapidly cooled to room temperature, and added 200. mu.L of 1 mg. mL-1The absorbance of the vanillin solution (prepared from a 17% phosphoric acid solution) is measured at 522nm wavelength after the dark reaction is carried out for 4-15 min at normal temperature, as shown in figure 2, the result indicates that the absorbance of the derivative product of the lipid and the vanillin is firstly increased and then reduced along with the increase of the derivatization reaction time, and the measured absorbance reaches the highest value when the dark reaction is carried out for 8min, preferably, the condition is that the total lipid content in the sample is in direct proportion to the absorbance in the method for measuring the total lipid content in the sample. The total lipid content of the sample was determined according to the preferred total lipid assay method and the results are illustrated in FIG. 3: the lipid content in the samples treated with methyl t-butyl ether was significantly reduced, but the samples treated with perchloric acid had very little lipid content. And (4) prompting by a result: the acetonitrile and the methyl tert-butyl ether can be used for treating the sample simultaneously, so that the lipid content in the sample can be obviously reduced, and the protein and lipid content in the sample are smaller when the sample is treated by the perchloric acid alone.
The amino acids in the treated samples were determined by HPLC after derivatization with o-phthalaldehyde. The liquid phase conditions include: waterse2695 separating Module-2475FLRDetector-2998PDADetector, chromatographic column: ODSC18 (250X 4.6mm, 5 μm), mobile phase A methanol and mobile phase B30 mmol. multidot.L-1The sodium acetate buffer solution (2) having a pH of 6.20 was sampled in an amount of 20. mu.L at a flow rate of 0.9 mL. min-1The column temperature was 30 ℃ and the analysis time was 56 min. The excitation wavelengths of the fluorescence detector are: 340nm, emission wavelength: 450nm, and the detection wavelength of the PDA detector is 230 nm. Figures 4, 5, 6 illustrate: in the chromatogram when the wavelength of the perchloric acid and the methyl tert-butyl ether + perchloric acid treatment sample is set as 230nm by an ultraviolet detector, the chromatogram has no obvious difference; in chromatograms of acetonitrile, methyl tert-butyl ether and acetonitrile treated samples when the wavelength of an ultraviolet detector is set to be 230nm, the chromatograms have no obvious difference; in the chromatogram of the acetonitrile and perchloric acid-treated sample at the wavelength of the ultraviolet detector set to 230nm, the impurities in the acetonitrile sample are more,especially the retention time is in the range of 0-10 min; HPLC ultraviolet detection chromatogram results show that the perchloric acid-treated sample has fewer impurities.
The evaluation result of the pretreatment method indicates that: preferably, the sample is treated with perchloric acid alone to reduce the protein and lipid content of the sample.
Case three, determining the pretreatment conditions of the sample
The preferable result selection pretreatment method directly adopts 0.4 mol.L-1The perchloric acid solution of (a) removes lipids and proteins from the sample. In order to further optimize the sample pretreatment method, the consumption of perchloric acid is optimized. Sample 200 μ L, as per 1: 3-1: to 9 (sample: perchloric acid solution ═ μ L:. mu.l) was added 0.4 mol. mu.l-1The perchloric acid solution was vortexed for 30 seconds, centrifuged at 15000g at 4 ℃ for 15min, and the supernatant was collected. 0.4 mol.L according to different proportions-1The perchloric acid treated samples were examined for protein and lipid removal as in case two, and FIG. 7 illustrates: as the amount of perchloric acid used increases, the amount of protein removed increases, and in sample volume: the volume of the perchloric acid solution is 1: at 9, the amount of protein removed from the sample was the greatest. FIG. 8 illustrates: the amount of lipid removed increases with increasing amounts of perchloric acid. Sample volume: the volume of the perchloric acid solution is 1: in case of 9, the amount of protein and lipid removed was the largest in the direct precipitation method.
Directly adopting 0.4 mol.L-1The perchloric acid solution of (a) removes lipid and protein from the sample, and is derivatized with o-phthalaldehyde and then measured by HPLC. The liquid phase conditions include: waterse2695 separating Module-2475FLRDetector-2998PDADetector, chromatographic column: ODSC18 (250X 4.6mm, 5 μm), mobile phase A methanol and mobile phase B30 mmol. multidot.L-1The sodium acetate buffer solution (2) having a pH of 6.20 was sampled in an amount of 20. mu.L at a flow rate of 0.9 mL. min-1The column temperature was 30 ℃ and the analysis time was 56 min. The excitation wavelengths of the fluorescence detector are: 340nm, emission wavelength: 450 nm. The extraction amount of 18 amino acids was calculated from the peak area automatically integrated by the Empower software of Waters. As shown in fig. 9: sample volume: the volume of the perchloric acid solution is 1: 9, the direct precipitation method maximizes the extraction of 18 amino acids.
From the viewpoint of the amount of protein precipitated, the amount of lipid removed, and the amount of amino acid extracted, the preferred sample volume is: the volume of the perchloric acid solution is 1: 9 the direct precipitation method is the sample pretreatment condition.
Claims (2)
1. A pretreatment method for measuring 18 amino acids in brain tissues and plasma of a C57 mouse is characterized by comprising the following steps:
first, brain homogenate and plasma are prepared
Precisely weighing the brain tissue of a C57 mouse, and performing the following steps of 1: 5-1: 20 (the weight of the brain tissue is as high as mg and muL), adding ultrapure water, homogenizing for 30-90 s by using a handheld homogenizer to obtain a brain tissue homogenate, centrifuging a sample obtained by blood sampling from an orbit, and taking a supernatant to obtain plasma to be treated;
second step, sample treatment
Taking 100-300 mu L of sample, adding 100-300 mu L of methyl tert-butyl ether, performing vortex oscillation, centrifuging and discarding the upper layer to remove lipid in the sample; taking 100-300 mu L of the above degreased sample, adding 100-300 mu L of protein precipitator, and removing protein in the sample by vortex oscillation, centrifugation and supernatant taking;
third step, investigating total protein and total lipid content after sample treatment
Taking 100-300 mul of treated sample, measuring the absorbance of the sample at 280nm by using an ultraviolet spectrophotometer, and inspecting the total protein content in the treated brain tissue sample; taking 100-300 mul of the treated sample in a glass test tube, volatilizing the solvent, adding 200 mul of concentrated H2SO4(96%), vortex, heat in 90 deg.C water bath for 20min, cool to room temperature quickly, add 200. mu.L 1 mg. mL-1Vanillin solution (prepared by 17% phosphoric acid solution), reacting at normal temperature in dark place, and measuring absorbance at 500-550 nm wavelength;
fourthly, the HPLC diode array detector switches the fluorescence detector to investigate the impurity removal amount and the amino acid extraction amount
The liquid phase conditions of HPLC comprise chromatographic column, mobile phase A and mobile phase B, wherein the chromatographic column is octadecaneSilane bonding silica gel column, mobile phase A is methanol, mobile phase B is 30 mmol.L-1And sodium acetate buffer solution with pH of 6.20, the amino acids in brain tissue and blood plasma samples were measured after derivatization with o-phthalaldehyde at a flow rate of 0.9 mL/min and a sample size of 20 μ L-1The column temperature is 30 ℃, the detection wavelength of the PDA detector is 230nm, the excitation wavelength of the fluorescence detector is 340nm, and the emission wavelength is 450 nm;
fifthly, determining the pretreatment condition of the sample
The result selection pretreatment method in the fourth step directly adopts 0.1-0.5 mol.L-1The perchloric acid solution removes lipid and protein in the sample, optimizes the dosage of perchloric acid for further optimizing the sample pretreatment method, and adopts the following steps of 1: 3-1: 9 (sample: perchloric acid solution ═ muL:. mu.L) ratio of 0.1 to 0.5 mol. mu.L-1Optimizing the sample after the perchloric acid treatment to obtain a sample pretreatment method through investigation of the content of total protein and total lipid;
2. the samples described herein are C57 mouse cortex and plasma, the 18 amino acids are: glutathione (Gsh), aspartic acid (Asp), glutamic acid (Glu), serine (Ser), glutamine (Gln), citrulline (Cit), glycine (Gly), arginine (Arg), taurine (Tau), tyrosine (Tyr), alanine (Ala), gamma-aminobutyric acid (GABA), tryptophan (Trp), methionine (Met), valine (Val), phenylalanine (Phe), isoleucine (Ile), leucine (Leu).
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