CN112180013B - Intestinal microbial metabolism marker composition for myocardial infarction diagnosis and detection method and application thereof - Google Patents
Intestinal microbial metabolism marker composition for myocardial infarction diagnosis and detection method and application thereof Download PDFInfo
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- CN112180013B CN112180013B CN202011050473.4A CN202011050473A CN112180013B CN 112180013 B CN112180013 B CN 112180013B CN 202011050473 A CN202011050473 A CN 202011050473A CN 112180013 B CN112180013 B CN 112180013B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
Abstract
The invention provides an intestinal microorganism metabolism marker composition for diagnosing myocardial infarction, which comprises one or more of acetyl L-carnitine, gamma-butyl betaine and choline chloride. Preferably, the intestinal microbial metabolism marker composition for diagnosing myocardial infarction comprises choline chloride and acetyl-L-carnitine. The intestinal microbial metabolism marker composition for diagnosing myocardial infarction comprises choline chloride and gamma-butyl betaine. The intestinal microbial metabolism marker composition for diagnosing myocardial infarction comprises choline chloride, acetyl-L-carnitine and gamma-butyl betaine. Related detection methods and applications are also provided. The intestinal microorganism metabolism marker composition for diagnosing the myocardial infarction can be used for diagnosing the myocardial infarction, can accurately, quickly and highly specifically diagnose the myocardial infarction with high sensitivity, provides guarantee for early treatment as soon as possible, and is suitable for large-scale popularization and application.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to the technical field of myocardial infarction diagnosis markers, and specifically relates to an intestinal microbial metabolism marker composition for myocardial infarction diagnosis, and a detection method and application thereof.
Background
Cardiovascular diseases are the leading cause of death of urban and rural residents in China at present, and are higher than tumors and other diseases. The newly released data of 'Chinese cardiovascular disease report 2018' show that: the morbidity and mortality of cardiovascular diseases in China are still in a continuous rising stage, 2.9 million patients with cardiovascular diseases are calculated, wherein 1300 million stroke, 1100 million coronary heart disease, 500 million pulmonary heart disease, 45 million heart failure, 250 million rheumatic heart disease and 2.7 million hypertension exist in the whole country, the cardiovascular diseases are main killers of human health, the burden of the cardiovascular diseases is gradually increased, the cardiovascular diseases become a great public health problem, and the prevention and the treatment of the cardiovascular diseases are not easy enough. How to early prevent and treat cardiovascular diseases, find effective targets of drug action, open up new therapeutic approaches, and become a hot spot of attention.
In China, 1100 thousands of people have coronary heart disease patients, and the coronary heart disease comprises angina, myocardial infarction, sudden death, arrhythmia, heart failure and the like. Myocardial infarction is a very serious type in coronary heart disease, and once myocardial infarction occurs, the myocardial infarction indicates that the blood vessel has acute and complete occlusion, and is possibly life-threatening or even causes sudden death. Myocardial infarction is the most common critical illness in the chest pain center of hospitals, the clinical diagnosis is mainly carried out by comprehensive analysis according to several aspects of medical history, electrocardiogram, change of serum enzyme and the like, and biomarkers are widely used for diagnosis, clinical evaluation, prognosis evaluation and the like of myocardial infarction.
Currently, the detection of biomarkers for cardiovascular and cerebrovascular diseases is mainly focused on macromolecules, such as troponin, CK-MB, myoglobin and cardiac fatty acid binding protein, which are markers of myocardial injury; markers of risk factors for coronary artery disease, such as TC, LDL-C, HDL-C, lpa, TG, CRP, LP-PLA2, and the like; a thromboembolic biomarker D-dimer; b-type natriuretic peptide (BNP) or N-terminal pro-B-type natriuretic peptide (NT-proBNP) as biomarkers for diagnosis, evaluation of therapeutic effect, and prognosis of heart failure. The existing detection methods for macromolecules mostly adopt biochemical methods, immunological methods and the like, and the methods have low specificity and accuracy and cannot completely meet higher clinical requirements. Some biomarkers, such as NT-proBNP and BNP, may have certain effects on the results due to the influence of demographic characteristics, sample storage conditions (the markers are active and easy to decompose), and drugs. In particular, there is a lack of biomarkers that can early warn of cardiovascular events.
The human intestinal flora is a complex community, and the intestinal microbiota plays an important role in immunity and defense, digestion and metabolism, inflammation, and cell proliferation. The main nutrients choline, betaine and carnitine from red meat, eggs, dairy products and saltwater fish are involved in biological activities such as energy metabolism in human bodies. After ingestion, fermentation of these nutrients by gut microbes results in the release of Trimethylamine (TMA), which is converted to trimethylamine oxide (TMAO) by the host liver enzymes, flavin-containing monooxygenase 3 (FMO 3). There is increasing evidence that: TMAO, one of small molecules in intestinal microorganism metabolism, participates in cholesterol metabolism, promotes platelet high aggregation, increases thrombus formation, and promotes vascular inflammatory reaction to cause arterial plaque formation. However, no metabolic markers relevant to diagnosis of cardiovascular diseases (particularly myocardial infarction) exist at present.
Therefore, there is an urgent need in the art to find more intestinal microbial metabolic markers on the "intestinal mandrel" related to diagnosis and early warning of cardiovascular diseases (especially myocardial infarction) by using LC-MS/MS detection platform and detecting one or more intestinal microbial metabolic markers with higher stability, specificity, sensitivity and accuracy by using large molecules.
Disclosure of Invention
In order to overcome the defects in the prior art, an object of the present invention is to provide an intestinal microbial metabolism marker composition for diagnosing myocardial infarction, which can be used for diagnosing myocardial infarction, can accurately, quickly, specifically and sensitively diagnose myocardial infarction, provides a guarantee for early treatment as soon as possible, and is suitable for large-scale popularization and application.
Another object of the present invention is to provide a method for detecting an intestinal microbial metabolism marker composition for diagnosing myocardial infarction, which can detect the content of the intestinal microbial metabolism marker composition for diagnosing myocardial infarction, so that the method can be used for diagnosing myocardial infarction, can accurately, rapidly, highly specifically and highly sensitively diagnose myocardial infarction, provide a guarantee for early treatment as soon as possible, and is suitable for large-scale popularization and application.
Another object of the present invention is to provide an application of the intestinal microorganism metabolism marker composition for diagnosing myocardial infarction, which can be used for diagnosing myocardial infarction, can accurately, rapidly, highly specifically and highly sensitively diagnose myocardial infarction, provides a guarantee for early treatment as soon as possible, and is suitable for large-scale popularization and application.
To achieve the above objects, in a first aspect of the present invention, there is provided an intestinal microbial metabolism marker composition for diagnosing myocardial infarction, characterized by comprising one or more of acetyl-l-carnitine, gamma-butylbetaine, and choline chloride.
Preferably, the intestinal microbial metabolism marker composition for diagnosing myocardial infarction comprises choline chloride and acetyl-L-carnitine.
Preferably, the intestinal microbial metabolism marker composition for diagnosing the myocardial infarction comprises choline chloride and gamma-butyl betaine.
Preferably, the intestinal microbial metabolism marker composition for diagnosing the myocardial infarction comprises choline chloride, acetyl-L-carnitine and gamma-butyl betaine.
In a second aspect of the present invention, there is provided a method for detecting the content of the above-mentioned intestinal microbial metabolism marker composition for diagnosing myocardial infarction in a biological sample, wherein the method employs a high performance liquid chromatography-mass spectrometry method, and the method comprises the following steps:
(1) Adding an internal standard of the intestinal microbial metabolism marker composition for diagnosing the myocardial infarction into a biological sample to be detected, adding an extraction solution, mixing, centrifuging and taking a supernatant, wherein the extraction solution is used for extracting the intestinal microbial metabolism marker composition for diagnosing the myocardial infarction and the internal standard from the biological sample to be detected;
(2) And (2) determining the content of the intestinal microbial metabolism marker composition for diagnosing the myocardial infarction in the biological sample to be detected by using a liquid chromatograph-mass spectrometer:
A. passing the supernatant through high performance liquid chromatography of the LC-MS to obtain HPLC eluent;
B. ionizing the HPLC eluate by an electrospray ion source of the mass spectrum of the LC-MS to respectively generate precursor ions of the intestinal microbial metabolism marker composition for myocardial infarction diagnosis and precursor ions of the internal standard;
C. generating fragment ions of the precursor ions of the intestinal microorganism metabolism marker composition for myocardial infarction diagnosis and fragment ions of the precursor ions of the internal standard respectively;
D. comparing the content of the precursor ions in the step B with the content of the fragment ions in the step C so as to determine the content of the intestinal microbial metabolism marker composition for diagnosing myocardial infarction in the biological sample to be detected.
Preferably, the extraction solution is an acetonitrile solution containing 0.5 vol% to 2.0 vol% acetic acid.
More preferably, the acetic acid is a 1.0% to 1.5% by volume solution of acetic acid in acetonitrile.
Preferably, the biological sample to be detected is selected from one or more of blood, serum and plasma.
In a third aspect of the present invention, there is provided a use of the above intestinal microbial metabolism marker composition for diagnosing myocardial infarction as a reagent for diagnosing myocardial infarction.
In a fourth aspect of the present invention, there is provided an application of the above intestinal microbial metabolism marker composition for diagnosing myocardial infarction in the preparation of a myocardial infarction diagnostic kit.
The invention has the beneficial effects that:
1. the intestinal microbial metabolism marker composition for diagnosing the myocardial infarction comprises one or more of acetyl L-carnitine, gamma-butyl betaine and choline chloride, and the AUC is over 0.8 when the intestinal microbial metabolism marker composition for diagnosing the myocardial infarction is used for diagnosing and distinguishing myocardial infarction patients from healthy people, so that the intestinal microbial metabolism marker composition can be used for diagnosing the myocardial infarction, can accurately, quickly, highly specifically and highly sensitively diagnose the myocardial infarction, provides guarantee for early treatment as soon as possible, and is suitable for large-scale popularization and application.
2. The method for detecting the content of the intestinal microorganism metabolism marker composition for diagnosing the myocardial infarction in the biological sample adopts a high performance liquid chromatography-mass spectrometry combined method, and comprises the following steps of: adding an internal standard into a biological sample to be detected, adding an extraction solution, mixing, centrifuging and taking a supernatant; and (3) determining the content of the intestinal microbial metabolism marker composition for diagnosing the myocardial infarction by using a liquid chromatograph-mass spectrometer: A. passing the supernatant through HPLC of LC-MS to obtain HPLC eluate; B. ionizing the HPLC eluent by an electrospray ion source of the mass spectrum of the liquid chromatograph-mass spectrometer to respectively generate an intestinal microbial metabolism marker composition and an internal standard precursor ion for diagnosing the myocardial infarction; C. generating fragment ions of the precursor ions, respectively; D. and C, comparing the content of the precursor ions in the step B with the content of the fragment ions in the step C to determine the content of the intestinal microbial metabolism marker composition for diagnosing the myocardial infarction in the biological sample to be detected, so that the content of the intestinal microbial metabolism marker composition for diagnosing the myocardial infarction can be detected, the myocardial infarction can be diagnosed accurately, quickly, highly specifically and highly sensitively, a guarantee is provided for early treatment as soon as possible, and the kit is suitable for large-scale popularization and application.
3. The intestinal microorganism metabolism marker composition for diagnosing the myocardial infarction is applied to being used as a myocardial infarction diagnosis reagent and being used for preparing a myocardial infarction diagnosis kit, so that the intestinal microorganism metabolism marker composition can be used for diagnosing the myocardial infarction, can accurately, quickly, highly specifically and sensitively diagnose the myocardial infarction, provides guarantee for early treatment as soon as possible, and is suitable for large-scale popularization and application.
These and other objects, features and advantages of the present invention will become more fully apparent from the following detailed description, the accompanying drawings and the appended claims, wherein like reference numerals refer to like parts throughout the several views, and wherein like reference numerals refer to like parts throughout the several views.
Drawings
Fig. 1 is a standard regression curve of the acetyl l-carnitine standard solution obtained in example 1, wherein the compound name: acetyl L-carnitine, m/z:204.00> < 85.10, standard curve formula:
f (x) =2.56170 x-0.0121034, correlation coefficient (R) =0.9990375, and fitting degree (R ^ 2) =0.9980759.
FIG. 2 is a standard regression curve of the betaine standard solution obtained in example 1, wherein the compound name: betaine, m/z:118.10 > 58.10, standard curve formula: f (x) =1.36364 x +7.45815, correlation coefficient (R) =0.9999555, and fitting degree (R ^ 2) =0.9999109.
Fig. 3 is a standard regression curve of the choline chloride standard solution obtained in example 1, wherein the compound name: choline chloride, m/z:104.15>, 85.10, standard curve formula:
f (x) =1.50986 x +0.0657501, correlation coefficient (R) =0.9950757, fitting degree (R ^ 2) =0.9901756.
FIG. 4 is a standard regression curve of the crotonobetaine standard solution obtained in example 1, wherein the compound name: crotonobetaine, m/z:144.00 > 58.10, standard curve formula:
f (x) =1.76295 x-0.00474892, correlation coefficient (R) =0.9966557, and fitting degree (R ^ 2) =0.9933226.
Fig. 5 is a standard regression curve of the gamma-butyl betaine standard solution obtained in example 1, wherein the compound name: γ -butylbetaine, m/z:146.00 >' 87.05, standard curve formula:
f (x) =1.33469 x +0.0430853, correlation coefficient (R) =0.9991281, fitting degree (R ^ 2) =0.9982570.
Fig. 6 is a standard regression curve of the l-carnitine standard solution obtained in example 1, wherein the compound name: l-carnitine, m/z:162.10>, 60.10, standard curve formula: f (x) =1.22396 x +0.101984, correlation coefficient (R) =0.9982382, fitting degree (R ^ 2) =0.9964796.
Fig. 7 is a standard regression curve of the trimethyllysine standard solution obtained in example 1, wherein the compound name: trimethyllysine, m/z:189.20 > 84.10, standard curve formula: f (x) =1.14304 x +0.0849619, correlation coefficient (R) =0.9991111, and fitting degree (R ^ 2) =0.9982230.
Fig. 8 is a standard regression curve of the trimethylamine oxide standard solution obtained in example 1, wherein the compound name: trimethylamine oxide, m/z:76.15>, 58.15, standard curve formula:
f (x) =0.992700 x +0.0552546, correlation coefficient (R) =0.9975928, and fitting degree (R ^ 2) =0.9951915.
FIG. 9 is a diagram of a Receiver Operating Curve (ROC) model for index joint test using the ROC method.
Detailed Description
The present inventors have extensively and intensively studied to provide a biomarker composition capable of being accurately diagnosed for myocardial infarction through a large number of screens and tests. The invention finds that choline chloride can be used as a biomarker for diagnosing myocardial infarction, and the invention can diagnose whether a patient suffers from myocardial infarction with high specificity, high sensitivity and high accuracy by jointly applying the choline chloride and the acetyl-L-carnitine, jointly applying the choline chloride and the gamma-butyl betaine and by using the choline chloride, the acetyl-L-carnitine and the gamma-butyl betaine, and reduces the misdiagnosis rate.
Term(s) for
As used herein, the terms "comprising," "including," "containing," and "containing" are used interchangeably and include not only closed-form definitions, but also semi-closed and open-form definitions. In other words, the term includes "consisting of 8230; \8230; composition;" consisting essentially of 8230; \8230; composition 8230).
As used herein, the term "liquid-mass spectrometry" is short for high performance liquid-mass spectrometry, i.e., "liquid-mass spectrometry" is used interchangeably with "high performance liquid-mass spectrometry".
As used herein, the term "acetyl-l-carnitine" is abbreviated ALC, i.e. "acetyl-l-carnitine" is used interchangeably with ALC.
As used herein, the term "trimethylamine oxide" is abbreviated TMAO, i.e., "trimethylamine oxide" and "TMAO" are used interchangeably.
As used herein, the term "ultra performance liquid chromatography" is abbreviated UPLC, i.e., "ultra performance liquid chromatography" and "UPLC" are used interchangeably.
In the present invention, it is understood that the mobile phase flow rate is the sum of the flow rates of the mobile phase a and the mobile phase B.
As used herein, the term "acetyl-l-carnitine-D3" and "ALC-D3" are used interchangeably to refer to a deuteron formed after H in acetyl-l-carnitine is substituted with deuterium isotope, which can be used as an internal standard for mass spectrometric detection of acetyl-l-carnitine in the present invention.
As used herein, the term "trimethylamine oxide-D9" is used interchangeably with "TMAO-D9" and in the present invention is used as an internal standard for the mass spectrometric detection of trimethylamine oxide and trimethyllysine.
As used herein, the terms "fragment ion" and "daughter ion" are used interchangeably and refer to those resulting from a single molecular fragmentation reaction of a molecular ion or larger fragment ion.
As used herein, the terms "mobile phase A", "phase A" or "aqueous mobile phase" are used interchangeably and refer to the mobile phase A obtained by adding the mobile phase additive A and the mobile phase additive B to ultrapure water in the liquid phase condition in a ratio of 0.05-0.4% (v/v) formic acid and 0.01-0.1% (v/v) ammonia water, respectively.
As used herein, the terms "mobile phase B", "B phase" or "organic mobile phase" are used interchangeably to refer to the condition of a liquid phase wherein said mobile phase B comprises 0.05-0.4% (v/v) formic acid in acetonitrile.
As used herein, the term "HPLC" or "high performance liquid chromatography" refers to liquid chromatography in which the degree of separation is increased by passing a mobile phase under pressure through a stationary phase, typically a tightly packed column.
As used herein, "mass spectrometry" (MS) refers to analytical techniques for identifying compounds by their mass MS techniques generally include (1) ionizing a compound to form a charged compound; and (2) detecting the molecular weight of the charged compound and calculating the mass to charge ratio (m/z), the compound being ionized and detected by any suitable means, and "mass spectrometers" generally include ionizers and ion detectors.
The term "about" as used herein in reference to a quantitative measurement means that the indicated value is plus or minus 10%.
Detection method
The invention also provides a detection method for detecting various intestinal microbial metabolites represented by acetyl L-carnitine, betaine, choline chloride, crotonobetaine hydrochloride, gamma-butyl betaine, L-carnitine, trimethyl lysine and trimethylamine oxide.
In general, the method of the invention comprises the steps of:
(i) Ionizing said one or more intestinal microbial metabolites and an internal standard by an electrospray ion source (ESI) to produce at least one precursor ion of said one or more intestinal microbial metabolites and said internal standard, respectively;
(ii) (ii) generating one or more fragment ions of said precursor ion of said one or more intestinal microbial metabolites and said internal standard, respectively;
(iii) (iii) comparing the amount of the one or more intestinal microbial metabolites produced in steps (i) and (ii) with the amount of the one or more ions of the internal standard to determine the amount of the one or more intestinal microbial metabolites in the biological sample;
wherein, formic acid and ammonia water are added into a mobile phase in the chromatographic detection process.
The method is particularly suitable for detecting acetyl L-carnitine, betaine, choline chloride, crotonobetaine hydrochloride, gamma-butyl betaine, L-carnitine, trimethyl lysine and trimethylamine oxide.
In a preferred embodiment, the detection method of the present invention comprises the steps of:
(1) The standard substance, the internal standard substance and the extraction solvent are added into a quality control product and a blood sample to be detected, after full reaction, supernatant is taken, and the content of the intestinal flora metabolites in the blood sample to be detected is determined and analyzed through high performance liquid chromatography-mass spectrometry.
In a preferred embodiment, the chromatographic conditions of the high performance liquid phase are as follows:
a chromatographic column: a reverse phase chromatography column; and
mobile phase: phase A: water (v/v, based on the total volume of phase a) containing 0.05% to 0.4% formic acid (preferably 0.05% to 0.2%, more preferably 0.08% to 0.12%) and 0.01% to 0.1% ammonia (preferably 0.03% to 0.1%, more preferably 0.05% to 0.07%). And adding formic acid and ammonia water into the phase A for adjusting the peak shape of the chromatographic peak. The formic acid and the part of the ammonia water may also be added in the form of ammonium formate or the like.
Phase B: acetonitrile (v/v, based on the total volume of phase B) containing 0.05% to 0.4% formic acid (preferably 0.07% to 0.25%, more preferably 0.09% to 0.15%).
And (3) eluting a mobile phase: the mobile phase elution is gradient elution, and the procedure of the gradient elution is as follows:
preferably, the gradient elution procedure comprises:
in another preferred embodiment, the gradient elution procedure comprises:
in another preferred embodiment, the gradient elution procedure comprises:
in another preferred embodiment, the gradient elution procedure comprises:
in another preferred embodiment, the gradient elution procedure comprises:
0-1.5min: the mobile phase B accounts for 80-99 percent, and the balance is the mobile phase A;
1.5-2.5min: the mobile phase B is 35-90%, and the balance is the mobile phase A;
2.5-3.5min: the mobile phase B is 35-99%, and the balance is the mobile phase A;
3.5-5.0min: the mobile phase B is 92-99%, and the balance is the mobile phase A.
In another preferred embodiment, the high performance liquid further has one or more chromatographic conditions selected from the group consisting of:
temperature of the column: 25-50 ℃, preferably 35-45 ℃;
sample chamber temperature: 2-20 ℃, preferably 5-15 ℃;
flow rate of mobile phase (mobile phase a + mobile phase B): 0.1-1.5mL/min, preferably 0.2-1.0mL/min, more preferably 0.3-0.8mL/min, most preferably 0.4-0.6mL/min.
In another preferred embodiment, the reverse phase chromatographic column is an acquired UPLC BEH HILIC.
In another preferred embodiment, the reverse phase chromatographic column has a specification of (1.8-2.5) × (40-200) mm, 1.5-2.3. Mu.M). Typically, the reverse phase chromatography column is of size (2.1X 100mm, 1.7. Mu.M).
Typically, the reverse phase chromatography column is an ACQUITY UPLC BEH HILIC (2.1X 100mm, 1.7. Mu.M). Preferably, the chromatographic column is also provided with a protective pre-column, such as a protective pre-column: ACQUITY UPLC BEH HILIC VAnGuard pre-column (A), (B)
1.7. Mu.M, 2.1X 5 mm). The protective pre-column can intercept part of impurities in the sample, thereby protecting the chromatographic column.
In another preferred embodiment, the biological sample is selected from the group consisting of: blood, serum, plasma, or a combination thereof, preferably, the biological sample is peripheral whole blood.
Typically, in the mass spectrometry phase, the sample undergoes the following phases:
(i) Ionizing the HPLC eluate by an electrospray ionization source (ESI) to produce precursor ions of the biomarker and internal standard;
(ii) The precursor ions are collided within the mass spectrum, respectively producing fragment ions of the precursor ions.
In another preferred example, the mass spectrum is a tandem mass spectrum.
In another preferred example, the one or more intestinal microbial metabolites comprises acetyl-l-carnitine, and wherein the mass/charge ratio of the parent ion of the acetyl-l-carnitine is 204.00> 85.10;
the one or more intestinal microbial metabolites comprises betaine, and wherein the parent ion of the betaine has a mass/charge ratio of 118.10 > 58.10;
said one or more intestinal microbial metabolites comprises choline chloride, and wherein said parent ion of said choline chloride has a mass/charge ratio of 104.15> -85.10;
the one or more intestinal microbial metabolites comprise crotonobetaine hydrochloride, and wherein the parent ion mass/charge ratio of the crotonobetaine hydrochloride is 144.00 > 58.10;
the one or more gut microbial metabolites comprise gamma-butyl betaine, and wherein the parent ion of the gamma-butyl betaine has a mass/charge ratio of 146.00> 87.05;
the one or more intestinal microbial metabolites comprises l-carnitine, and wherein the mass/charge ratio of the parent ion of the l-carnitine is 162.10> 60.10;
said one or more intestinal microbial metabolites comprises trimethyllysine, and wherein said parent ion of said trimethyllysine has a mass/charge ratio of 189.20 > 84.10;
the one or more intestinal microbial metabolites comprises trimethylamine oxide, and wherein the mass/charge ratio of the parent ion of the trimethylamine oxide is 76.15> 58.15;
the methods of the invention are non-therapeutic and non-diagnostic in vitro.
Reagent kit
In the present invention, the kit of the present invention further comprises the set described below.
In another preferred embodiment, each biomarker in the collection according to the invention is used as a standard.
In another preferred embodiment, the kit further comprises an isotopic internal standard for the biomarker.
In another preferred embodiment, the kit further comprises an instruction describing a reference data set of levels (e.g., amounts) of the respective biomarkers from the myocardial infarction patient and/or the healthy control.
Preferably, the detection kit of the invention comprises:
(1) A standard, the standard comprising choline chloride;
(2) A quality control material, wherein the quality control material contains choline chloride;
(3) An isotope internal standard extracting solution containing choline chloride-D9 and methanol;
(4) An extraction solvent comprising component (i) acetonitrile and component (ii) acetic acid;
(5) A mobile phase additive a comprising formic acid; and
(6) A mobile phase additive B comprising ammonia.
In another preferred embodiment, the volume ratio (v/v) of the component (ii) in the extraction solvent is 0.5 to 2.0%, preferably 1.0 to 1.5%.
In another preferred embodiment, the kit further comprises a component selected from the group consisting of:
96 well reaction plate, instructions.
In another preferred example, the detection kit further comprises (7) a reference substance, wherein the reference substance contains choline chloride.
Preferably, the detection kit of the invention comprises:
(1) A standard, wherein the standard contains choline chloride and acetyl L-carnitine;
(2) A quality control product, wherein the quality control product contains choline chloride and acetyl L-carnitine;
(3) An isotope internal standard extracting solution, wherein the isotope internal standard extracting solution contains choline chloride-D9, acetyl L-carnitine-D3 and methanol;
(4) An extraction solvent comprising component (i) acetonitrile and component (ii) acetic acid;
(5) A mobile phase additive a comprising formic acid; and
(6) A mobile phase additive B comprising ammonia.
In another preferred example, the concentrations of choline chloride-D9 and acetyl L-carnitine-D3 in the isotope internal standard extracting solution are the same.
In another preferred embodiment, the volume ratio (v/v) of the component (ii) in the extraction solvent is 0.5 to 2.0%, preferably 1.0 to 1.5%.
In another preferred embodiment, the kit further comprises a component selected from the group consisting of:
96 well reaction plate, instructions.
In another preferred example, the detection kit further comprises (7) a reference substance, wherein the reference substance contains choline chloride and acetyl L-carnitine.
Preferably, the detection kit of the invention comprises:
(1) A standard comprising choline chloride, gamma-butyl betaine;
(2) A quality control material containing choline chloride and gamma-butyl betaine;
(3) An isotope internal standard extracting solution containing choline chloride-D9, gamma-butyl betaine-D9 and methanol;
(4) An extraction solvent comprising component (i) acetonitrile and component (ii) acetic acid;
(5) A mobile phase additive A comprising formic acid; and
(6) A mobile phase additive B, wherein the mobile phase additive B comprises ammonia water.
In another preferred example, the concentrations of choline chloride-D9 and gamma-butyl betaine-D9 in the isotope internal standard extracting solution are the same.
In another preferred embodiment, the volume ratio (v/v) of the component (ii) in the extraction solvent is 0.5 to 2.0%, preferably 1.0 to 1.5%.
In another preferred embodiment, the kit further comprises a component selected from the group consisting of:
96 well reaction plate, instructions.
In another preferred example, the detection kit further comprises (7) a reference substance, wherein the reference substance contains choline chloride and gamma-butyl betaine.
Preferably, the detection kit of the invention comprises:
(1) A standard, wherein the standard contains choline chloride, acetyl L-carnitine and gamma-butyl betaine;
(2) A quality control product containing choline chloride, acetyl L-carnitine and gamma-butyl betaine; (ii) a
(3) An isotope internal standard extracting solution containing choline chloride-D9, acetyl L-carnitine-D3, gamma-butyl betaine-D9 and methanol;
(4) An extraction solvent comprising component (i) acetonitrile and component (ii) acetic acid;
(5) A mobile phase additive a comprising formic acid; and
(6) A mobile phase additive B comprising ammonia.
In another preferred example, the concentrations of choline chloride-D9, acetyl-L-carnitine-D3 and gamma-butyl betaine-D9 in the isotope internal standard extracting solution are the same.
In another preferred embodiment, the volume ratio (v/v) of the component (ii) in the extraction solvent is 0.5 to 2.0%, preferably 1.0 to 1.5%.
In another preferred embodiment, the kit further comprises a component selected from the group consisting of:
96 well reaction plate, instructions.
In another preferred example, the detection kit further comprises (7) a reference substance, wherein the reference substance contains choline chloride, acetyl-L-carnitine and gamma-butyl betaine.
ROC-AUC
ROC-AUC is a method for evaluating model accuracy, and an ROC curve is a coordinate graph formed by a Receiver operating characteristic curve (Receiver operating characteristic curve), a False positive probability (False positive rate) as a horizontal axis and a True positive probability (True positive rate) as a vertical axis, and is a comprehensive index reflecting continuous variables of sensitivity and specificity. AUC is the Area under the ROC curve (Area under the curve). The ROC-AUC value is between 1.0 and 0.5, the closer to 1, the better the diagnosis effect is, the lower the accuracy is at 0.5-0.7, the certain accuracy is at 0.7-0.9, and the higher the accuracy is at AUC above 0.9. AUC =0.5, indicating that the diagnostic method was completely ineffective and of no diagnostic value. AUC <0.5 does not fit the real situation, and rarely occurs in practice.
The main advantages of the invention include:
(a) The invention discloses a group of intestinal microbial metabolism markers for diagnosing myocardial infarction, which comprise one or more of acetyl L-carnitine, gamma-butyl betaine and choline chloride. Wherein, the acetyl L-carnitine is firstly applied to the diagnosis of myocardial infarction. The choline chloride in the intestinal microbial metabolism marker provided by the invention is verified to be more than 0.8 when being used for diagnosing and distinguishing myocardial infarction patients and healthy people (in an ROC curve evaluation method, the area value AUC under an ROC curve is more than 0.5 and is closer to 1, which shows that the diagnosis effect is better, the AUC has lower accuracy between 0.5 and 0.7, the AUC has certain accuracy between 0.7 and 0.9, and the AUC has higher accuracy above 0.9); when the choline chloride and the acetyl L-carnitine are jointly applied, the capacity of diagnosing and distinguishing myocardial infarction patients and healthy people can be obviously improved, the AUC is 0.923, and the diagnosis and distinguishing effect is good; when choline chloride and gamma-butyl betaine are jointly applied, the capacity of diagnosing and distinguishing myocardial infarction patients and healthy people can be obviously improved, the AUC is 0.845, and the diagnosis and distinguishing effect is good; when choline chloride, acetyl L-carnitine and gamma-butyl betaine are jointly applied, the AUC is 0.933, and the diagnosis and discrimination effects are better.
(b) The detection method is simple to operate, short in analysis time (joint inspection only needs 5 minutes), accurate in detection result and high in result reproduction rate (CV is less than 10%).
(c) The kit provided by the invention can be used for diagnosing myocardial infarction, improving the diagnosis convenience and promoting the standardization of a diagnosis method.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
Example 1: detection method and specific experimental process
1. Origin of specimen
After patient consent was obtained, plasma samples of 20 patients with myocardial infarction (troponin > 500 pg/mL) were collected, and 20 healthy persons were matched in age and sex with those with myocardial infarction, and blood sampling time was in the early morning and fasting state.
2. Main apparatus and equipment:
shimadzu LC-MS 8050; labsolutions instruments software.
3. Reagents and materials:
acetyl L-carnitine, betaine, choline chloride, crotonobetaine hydrochloride, gamma-butyl betaine, L-carnitine, trimethyl lysine and trimethylamine oxide standard products; acetyl L-carnitine-D3, gamma-butyl betaine-D9, betaine-D11, choline chloride-D9, L-carnitine-D9, trimethylamine oxide-D9 internal standard substances, methanol, acetonitrile, ethanol, formic acid, ammonia water and carbon adsorbed human plasma are all commercially available reagents.
4. Liquid chromatography and mass spectrometry conditions:
chromatographic conditions are as follows:
a chromatographic column: ACQUITY UPLC BEH HILIC (A), (B)
1.7μM，2.1×100mm)；
Protecting the pre-column: ACQUITY UPLC BEH HILIC VAnGuard pre-column (A), (B)
1.7μM，2.1×5mm)；
Mobile phase A:0.1% (v/v) formic acid (A) +0.025% (v/v) aqueous ammonia (B); and (3) mobile phase B:0.1% (v/v) formic acid (A) in acetonitrile; based on the total volume of the mobile phase B; flow rate 0.6mL/min: column temperature 45 ℃: sample chamber temperature: 8 ℃; the sample size is 1 mu L; needle washing liquid: 50% methanol water.
Mobile phase gradient method:
mass spectrum conditions:
detecting in positive ion MRM mode by adopting an electrospray ionization source (ESI);
| compound (I) | Monitoring ions | Deviation of Q1 Pre | Energy of collision | Deviation of Q3 Pre |
| Acetyl L-carnitine | 204.00＞85.10 | -14V | -22.0eV | -22V |
| Betaine | 118.10＞58.10 | -12V | -25.0eV | -21V |
| Choline chloride | 104.15＞58.10 | -11V | -31.0eV | -10V |
| Crotonobetaine | 144.00＞58.10 | -10V | -25.0eV | -10V |
| Gamma-butylbetaine | 146.00＞87.05 | -10V | -17.0eV | -16V |
| L-carnitine | 162.10＞60.10 | -11V | -17.0eV | -10V |
| Trimethyllysine | 189.20＞84.10 | -13V | -22.0eV | -15V |
| Oxetamine | 76.15＞58.15 | -12V | -22.0eV | -10V |
| Acetyl L-carnitine-D3 | 207.15＞85.05 | -10V | -22.0eV | -14V |
| Gamma-butylbetaine-D9 | 155.20＞87.00 | -15V | -20.0eV | -15V |
| betaine-D11 | 129.10＞66.15 | -14V | -30.0eV | -11V |
| Choline chloride-D9 | 113.05＞66.30 | -22V | -32.0eV | -25V |
| L-carnitine-D9 | 171.20＞69.20 | -12V | -19.0eV | -12V |
| TMAO-D9 | 85.15＞66.20 | -10V | -24.0eV | -12V |
The flow rate of atomized gas is 3L/min, and the flow rate of heated gas is 10L/min; the flow rate of the drying gas is 10L/min; interface temperature: 300 ℃; DL temperature: 300 ℃; temperature of the heating block: 400 ℃;
5. the experimental process comprises the following steps:
5-1 solution preparation
5-1-1 preparation of mixed standard curve solution:
accurately weighing appropriate amount of acetyl L-carnitine, betaine, choline chloride, crotonobetaine hydrochloride, gamma-butyl betaine, L-carnitine, trimethyl lysine and trimethylamine oxide standard substances, dissolving with 50% methanol aqueous solution, and respectively preparing into standard substance stock solutions with concentration of 5 mmol/L. Then, the standard substance stock solution with the concentration of 5mmol/L is sequentially diluted into the working solution of the following standard curve by 50 percent methanol aqueous solution.
Mixing standard substance solutions, wherein the concentrations of standard curves of the four substances, namely acetyl L-carnitine, crotonobetaine, gamma-butyl betaine and trimethyllysine, are 0.05, 0.25, 1, 2.5, 5, 10 and 25 mu mol/L; the concentrations of the standard curves of the four substances of the betaine, the choline chloride, the L-carnitine and the trimethylamine oxide are 0.1, 0.5, 2, 5, 10, 20 and 50 mu mol/L; the preparation matrix of the mixed standard solution is 50% methanol aqueous solution.
5-1-2 preparation of mixed internal standard solution:
accurately weighing a proper amount of acetyl L-carnitine-D3, gamma-butylbetaine-D9, betaine-D11, choline chloride-D9, L-carnitine-D9 and trimethylamine oxide-D9, dissolving the mixture by using methanol to obtain a 1mmol/L mixed internal standard storage solution, and diluting the mixed internal standard storage solution to a 10 mu mol/L mixed internal standard working solution by using methanol.
5-1-3 extraction solvent
The components of the extraction solvent are acetonitrile and acetic acid, and the volume percentage of the acetic acid is 1.25%.
5-2, preparation of a quality control product I and a quality control product II:
and (3) taking a proper amount of Seralab carbon to adsorb blank human plasma, adding a small amount of solution with the highest concentration point of the mixed standard substance, and respectively preparing a quality control I and a quality control II.
5-3, pretreatment and sample injection analysis of a plasma sample:
respectively adding 25 mu L of mixed standard working solution and 25 mu L of to-be-detected plasma sample into different wells of a 96-well plate, then sequentially adding 20 mu L of mixed internal standard into each well, then adding 400 mu L of acetic acid acetonitrile solution (the concentration of acetic acid is 1.25%), performing vortex for 1min, performing centrifugation at 4000rpm for 5min, taking 120 mu L of supernatant, respectively injecting 1 mu L of supernatant into a liquid chromatograph-mass spectrometer, and determining and analyzing the content (unit is mu M) of each metabolite of intestinal flora metabolites in the to-be-detected plasma sample. Wherein the standard regression curves of the above compounds are shown in FIGS. 1 to 8. As shown in FIGS. 1 to 8, the standard regression curves of the above compounds were very linear, R 2 Greater than 0.99, and meets the performance requirement.
5-4 results:
as shown in table 1 below, the sensitivity and specificity data automatically derived by ROC curve method specifically fixed all indexes to be 95% specific (i.e. only one of the healthy population samples is positive), and the AUC areas and thresholds obtained from the ROC curve are shown in table 1. In the ROC curve evaluation method, when the area value AUC under the ROC curve is greater than 0.5, the closer to 1, the better the diagnostic effect. AUC has lower accuracy at 0.5-0.7, certain accuracy at 0.7-0.9, and higher accuracy at 0.9 or above. Greater than the threshold is defined as + and less than the threshold is defined as-.
AUC and threshold value of each index in Table 1
Example 2: constructing ROC curve, and comparing the ability of the above intestinal flora metabolites alone or in combination to diagnose and differentiate patients with myocardial infarction from healthy people
The ability of the compound to diagnose myocardial infarction is judged by the expression levels of acetyl L-carnitine, betaine, choline chloride, crotonobetaine hydrochloride, gamma-butylbetaine, L-carnitine, trimethyl lysine and trimethylamine oxide in 20 samples of myocardial infarction patients and 20 samples of healthy people through verification by adopting a Receiver Operating Curve (ROC) method. The sensitivity and specificity data automatically derived from the ROC curve method, which fixes the specificity of all the indicators to 95% (i.e., only one positive sample in healthy human), are shown in table 2.
TABLE 2 ability of single differential metabolite diagnosis to differentiate myocardial infarction patients from healthy population
| Single differential metabolite | Specificity of | Sensitivity of the probe | AUC | Threshold value |
| Choline chloride | 95％ | 60％ | 0.805 | 109uM |
| Acetyl L-carnitine | 95％ | 30％ | 0.639 | 4.4uM |
| Gamma-butylbetaine | 95％ | 25％ | 0.687 | 1.90uM |
| L-carnitine | 95％ | 25％ | 0.637 | 123uM |
| Oxetamine | 95％ | 25％ | 0.486 | 4.4uM |
| Betaine | 95％ | 10％ | 0.523 | 104uM |
| Trimethyllysine | 95％ | 25％ | 0.517 | 9.36uM |
| Crotonobetaine | 95％ | 15％ | 0.274 | 5.5uM |
As can be seen from Table 2, choline chloride has a strong ability to differentiate patients with myocardial infarction from healthy people when used singly, and AUC is 0.805; the sensitivity is 60%; the specificity was 95%. Acetyl L-carnitine, gamma-butyl betaine, L-carnitine, trimethylamine oxide, betaine, trimethyl lysine and crotonobetaine have poor capability of being used for diagnosing and distinguishing myocardial infarction patients from healthy people, and the AUC is 0.639, 0.687, 0.637, 0.486, 0.523, 0.517 and 0.274 respectively; the sensitivities are respectively 30%, 25%, 10%, 25% and 15%; the specificity was 95%.
Further verifies whether the diagnosis effect is improved or not by the joint detection of any index of the choline chloride, the acetyl L-carnitine, the gamma-butyl betaine, the L-carnitine, the trimethylamine oxide, the betaine, the trimethyl lysine and the crotonobetaine.
TABLE 3 ability of two differential metabolite joint diagnosis to differentiate patients with myocardial infarction from healthy population
As shown in table 3, when any index of choline chloride, acetyl-l-carnitine, γ -butylbetaine, l-carnitine, trimethylamine oxide, betaine, trimethyllysine, and crotonobetaine is jointly detected and distinguished from myocardial infarction patients and healthy people, two groups of index combinations with better effect of diagnosing and distinguishing myocardial infarction patients and healthy people are selected from the index combinations.
When the choline chloride and the acetyl L-carnitine are jointly applied, the capacity of diagnosing and distinguishing myocardial infarction patients and healthy people can be obviously improved, the AUC is 0.923, and the diagnosis and distinguishing effect is good; when the specificity is 95%, the sensitivity is respectively improved from 30% and 60% in the single detection to 90% in the joint detection. When choline chloride and gamma-butyl betaine are used in combination, the AUC is 0.845, and the sensitivity is respectively improved from 25% and 60% in single detection to 85% in combined detection under the condition that the specificity is 95%.
TABLE 4 ability of the three differential metabolite joint diagnosis to differentiate patients with myocardial infarction from healthy population
| Three differential metabolite combinations | Specificity of | Sensitivity of the probe | AUC |
| Acetyl L-carnitine, gamma-butylbetaine and choline chloride | 95％ | 95％ | 0.933 |
In addition, as shown in table 4, when the combined application of three indexes, namely acetyl-l-carnitine, gamma-butyl betaine and choline chloride, is used for diagnosing and distinguishing myocardial infarction patients from healthy people, the AUC is closer to 1 than any two combined applications, the AUC is 0.933, and the diagnosis and distinguishing effect is the best; when the specificity is 95%, the sensitivity is respectively improved from 30%, 25% and 60% in the single detection to 95% in the joint detection. The ROC curve model diagram for index joint inspection refers to fig. 9, where reference numeral 1 denotes an ROC curve for index 1, reference numeral 2 denotes an ROC curve for index 2, reference numeral 3 denotes an ROC curve for index 1 and index 2 joint inspection, and reference numeral 3 denotes a reference line.
Example 3 preparation of assay kit
A detection kit is prepared based on the metabolic marker provided by the invention, and the kit comprises the following components: quality control product, isotope internal standard extracting solution, extracting solvent, mobile phase additive A and mobile phase additive B. Preferably, the test kit further comprises a control, a 96-well reaction plate, a 96-well filter plate, instructions, and the like. The kit can be used for detecting the content of three intestinal flora metabolites, namely choline chloride, acetyl L-carnitine and gamma-butyl betaine.
Specifically, in the kit of the invention, the reference substance and/or the quality control substance contains choline chloride, acetyl-L-carnitine and gamma-butyl betaine, the isotope internal standard extracting solution contains choline chloride-D9, acetyl-L-carnitine-D3 and gamma-butyl betaine-D9, the concentrations are all 5 μ M, the extracting solvent comprises a component (i) acetonitrile and a component (ii) acetic acid, the volume percentage (v/v) of the acetic acid is 1.25%, the mobile phase additive A is formic acid, and the mobile phase additive B is ammonia water. The kit is stored at 2-8 ℃.
Of course, when designing the detection kit, it is not necessary to completely contain the above-mentioned 3 metabolic markers as a standard, and only a few of them may be used in combination. The standard products can be packaged separately or made into mixture package. The kit is designed based on a plurality of metabolic markers provided by the invention, and can be used for diagnosing and distinguishing myocardial infarction patients from healthy people.
In conclusion, the present invention effectively overcomes the disadvantages of the prior art and has high industrial utilization value.
Therefore, the invention provides a group of intestinal microbial metabolism marker compositions for diagnosing myocardial infarction as well as a detection method and application thereof. Specifically, a biomarker set is provided, wherein the biomarker set comprises one or more of acetyl L-carnitine, gamma-butyl betaine and choline chloride. Wherein, the acetyl L-carnitine is firstly applied to the diagnosis of myocardial infarction. Proved by verification, when choline chloride in the intestinal microbial metabolism marker is used for diagnosing and distinguishing myocardial infarction patients from healthy people, the AUC is over 0.8; when the choline chloride and the acetyl L-carnitine are jointly applied, the capability of diagnosing and distinguishing myocardial infarction patients and healthy people can be obviously improved, the AUC is 0.923, and the diagnosis and distinguishing effect is good; when choline chloride and gamma-butyl betaine are jointly applied, the capacity of diagnosing and distinguishing myocardial infarction patients and healthy people can be obviously improved, the AUC is 0.845, and the diagnosis and distinguishing effect is good; when choline chloride, acetyl L-carnitine and gamma-butyl betaine are jointly applied, AUC is 0.933, and the diagnosis and differentiation effects are better. The myocardial infarction can be accurately diagnosed with high specificity and high sensitivity by detecting the biomarker set.
In conclusion, the intestinal microorganism metabolism marker composition for diagnosing myocardial infarction can be used for diagnosing myocardial infarction, can accurately, quickly, highly specifically and sensitively diagnose myocardial infarction, provides guarantee for early treatment as soon as possible, and is suitable for large-scale popularization and application.
It will thus be seen that the objects of the invention have been fully and effectively accomplished. The functional and structural principles of the present invention have been shown and described in the embodiments, and the embodiments may be modified without departing from the principles. Therefore, this invention includes all modifications encompassed within the spirit and scope of the claims.
Claims (8)
1. The application of the intestinal microbial metabolism marker composition for diagnosing the myocardial infarction in preparing a myocardial infarction diagnostic reagent, wherein the intestinal microbial metabolism marker composition for diagnosing the myocardial infarction comprises choline chloride.
2. The use of claim 1, wherein the intestinal microbial metabolism marker composition for the diagnosis of myocardial infarction comprises choline chloride and acetyl-L-carnitine.
3. The use of claim 1, wherein the gut microbial metabolism marker composition for the diagnosis of myocardial infarction comprises choline chloride and γ -butyl betaine.
4. The use of claim 1, wherein the intestinal microbial metabolism marker composition for the diagnosis of myocardial infarction comprises choline chloride, acetyl-l-carnitine and γ -butylbetaine.
5. The application of the intestinal microbial metabolism marker composition for diagnosing the myocardial infarction in preparing a myocardial infarction diagnosis kit is disclosed, wherein the intestinal microbial metabolism marker composition for diagnosing the myocardial infarction comprises choline chloride.
6. The use of claim 5, wherein the intestinal microbial metabolism marker composition for the diagnosis of myocardial infarction comprises choline chloride and acetyl-L-carnitine.
7. The use of claim 5, wherein the intestinal microbial metabolism marker composition for diagnosing myocardial infarction comprises choline chloride and gamma-butyl betaine.
8. The use of claim 5, wherein the intestinal microbial metabolism marker composition for the diagnosis of myocardial infarction comprises choline chloride, acetyl-L-carnitine and gamma-butylbetaine.
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