CN112630311A - Metabolic markers and kits for detecting affective disorders and methods of use - Google Patents
Metabolic markers and kits for detecting affective disorders and methods of use Download PDFInfo
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Abstract
The invention provides a metabolic marker for detecting affective disorder of a subject, comprising dopamine, 5-hydroxytryptamine, gamma-aminobutyric acid, tryptophan, tyramine, kynurenine, 3, 4-dihydroxyphenylacetic acid, epinephrine and norepinephrine; the affective disorder is selected from depression, schizophrenia and anxiety, and the depression is selected from dysthymia, endogenous depression, reactive depression, mild depression, major depression, psychotic depression, nervous depression, unipolar depression and bipolar depression. The invention also provides a kit for detecting the metabolic marker combination and a using method, and the kit has the advantages of convenience in use and reliable test result.
Description
Technical Field
The invention relates to the technical field of medical treatment, in particular to a group of metabolic markers capable of detecting depression of a subject, a kit containing the metabolic markers and diagnosis of affective disorder by detecting the metabolic markers. The invention further relates to a method for monitoring the effectiveness of a treatment for affective disorders.
Background
Depression, also known as depressive disorder, is characterized clinically by a marked and persistent depression in the mood, the main type of mood disorder. The low mood is not matched with the situation in clinic, the depression of the mood can be from sultriness to sadness, and the self-declining depression and even the pessimism are taken away, and suicide attempts or behaviors can be caused; even the occurrence of stupor; in some cases, there is significant anxiety and motor agitation; in severe cases, psychotic symptoms such as hallucinations and delusions may occur. Each episode lasts at least 2 weeks, more than long, or even years, and most cases have a tendency to have recurrent episodes, most of which can be alleviated, and some of which can have residual symptoms or become chronic. Depression is a common psychological disease, and by 2017, the number of people suffering from depression is more than 3.22 hundred million worldwide, and the prevalence rate is as high as 4.4 percent. Due to the particular psychological state of the depressed patients, and the limited level of clinical diagnosis, the recognition rate of depression is less than 20%. 40% of the annual suicidal population in our country is due to depression. However, the population of patients with depression at this stage is expanding in recessive and advancing towards a lower age.
Currently, the main criteria for the diagnosis of depression are the International Classification of diseases (ICD-10) and the diagnostic and statistical Manual of mental disorders (DSM-IV). ICD-10 is used as a standard in China. The difficulty in the diagnosis of depression is: 1. the disease image is atypical: like other mental disorders, the clinical signs of contemporary depression tend to be atypical. The typical "three low" symptoms (depressed mood, thought retardation and reduced motor behavior) are less common, which is associated with an expanded range of depression in the modern diagnostic classification and factors related to stress, personality and lifestyle habits of the patient. For example, some patients with depression may play computer games day by day and night, although they are said to have a lack of interest. Symptom hybridization: it can be seen that clinically, cases with difficult depression diagnosis are often mixed with many other mental symptoms, such as emotional irritations or dysthymia, obsessive-compulsive symptoms, anxiety symptoms, etc. Therefore, it is difficult to identify the diseases such as schizophrenia, obsessive-compulsive disorder and anxiety disorder. 3. The contradiction between standardized diagnosis and clinical practice: the diagnosis of schizoaffective disorder is reflected prominently, and some cases have a history of a plurality of typical schizophrenia and depression attacks, and the intermittence of the attacks is good. Since these symptoms occur in different stages, the diagnosis conditions of schizoaffective disorder are not satisfied with the current diagnosis standards, and the environmental and severe mood are different from the conventional diagnosis concept, so that many diagnosis and treatment problems need to be discussed. These diagnostic techniques are also highly dependent on subjective judgment by the doctor, and sometimes hidden or kneaded by the patient during the diagnosis. These conditions lead to difficulties in diagnosing depression and make the treatment of depression challenging.
The poor recognition rate of medical systems is mainly due to the clinical diagnosis of depression, which is usually done by means of questionnaires and inquiries. Such diagnostic means is too dependent on the judgment of the doctor, has very high requirements on the clinical experience of the doctor, and is not beneficial to large-scale popularization and screening. By combining the general phenomenon of low clinical diagnosis rate of depression patients, if an objective detection item is used as a screening tool of depression in the health management field for the prevention and treatment of depression, the 'line of defense' of depression can be advanced, a large number of potential patients with depression can be actively discovered, and the existing judgment means for the over-subjective clinical diagnosis of depression can be effectively supplemented. Only by carrying out active mental health management in the field of health physical examination and detecting and screening the objective physiological indexes of the depression, the early discovery and early intervention of the depression can be realized, and the key point of depression prevention and treatment is realized. Therefore, the development of a rapid, sensitive and accurate depression prediction and early diagnosis method has important significance for promoting the prevention and treatment work of depression in China.
There is no exact theory as to the origin and pathogenesis of depression, and environmental and physiological factors can influence it. It is now well recognized that depression may be caused by a deficiency or dysfunction of neurotransmitters, such as 5-hydroxytryptamine, epinephrine, etc., in the brain. Therefore, the metabolic level of neurotransmitters in an organism can reflect to some extent whether depression is present, such as dopamine, epinephrine, serotonin and gamma-aminobutyric acid (GABA). The compounds can be used as diagnostic biomarkers, and auxiliary early diagnosis of depression is carried out according to the in vivo metabolic level of a specific compound, so that mild patients or people with depression tendency are found at early stage, early intervention is carried out, and harm caused by depression is reduced. The ICD-10-based depression diagnosis method in China and abroad greatly depends on the subjective judgment of doctors, so that the depression is difficult to diagnose and the treatment of the depression is difficult.
In patent application No. CN201610105047.3 and patent application No. CN201080046087.6, the diagnosis of depression using serum metabolic markers is disclosed. However, the serum sample is difficult to collect and can be carried out by professional medical personnel, and the serum sample is difficult to be collected by the patient himself at home. In patent application No. CN201480019392.4, a diagnosis using serum or urine biomarkers is disclosed, the set of biomarkers used including at least cGMP, cortisol, calprotectin, thromboxane, aldosterone, HVEM and substance P. Because the used markers comprise small molecule metabolites and large molecule proteins, a plurality of technical platforms, such as mass spectrometry and immunological methods, are required to be used for detection, and the detection difficulty and cost are increased. And the invention uses serum and urine samples, the storage and transportation costs are high.
The literature (doctor university of Chongqing academic paper, authors: Zhenpeng, screening of diagnostic markers for plasma and urine of depression based on a metabonomic strategy, 2013 and Zheng P, Wang Y, Chen L, et al, identification and identification of effective metabolites for major depression disorder. mol Cell microorganisms.2013; 12(1): 207-. The AUC value for the 82 depression of the training set distinguishable from the 82 normal controls by this biomarker panel was 0.812 (95% confidence interval: 0.829-0.961); the AUC value distinguishing 44 depression from 52 normal controls in the test set was 0.895 (95% confidence interval: 0.829-0.961).
Traditional analytical methods such as immunoassay have been the most widely used techniques in clinical diagnosis, and in immunoassay, radioimmunoassay, the radioimmunoassay has been largely eliminated due to the presence of radioactive contamination. The ELISA method is manually operated, and the result is easily influenced by human factors. Due to the demands for detection convenience and detection result accuracy, development of new detection markers, detection methods, and detection tools is required.
Disclosure of Invention
One of the objectives of the present invention is to provide a group of metabolic markers that can be used for diagnosing affective disorders in a subject and for monitoring the treatment effect during treatment, and for evaluating the treatment effect of drugs for treating affective disorders.
It is a further object of the present invention to provide the use of said metabolic marker for diagnosing affective disorders in a subject, for the preparation of a reagent and a kit for detecting said metabolic marker.
It is a further object of the present invention to provide a method for detecting the type and amount of metabolic markers in a urine sample from a subject, more particularly a processed urine sample.
In the present invention, "affective disorder" is selected from depression, schizophrenia and anxiety, preferably depression; the depression is selected from dysthymia, endogenous depression, reactive depression, mild depression, major depression, psychotic depression, nervous depression, unipolar depression and bipolar depression. For the sake of brevity, the rest of the present invention is expressed as "depression", and those skilled in the art will understand that the principles and objectives of the described embodiments apply equally to other types of affective disorders.
As used herein, a "metabolic marker" refers to a unique biological or biogenic indicator of a process, event or disorder. Biomarkers can be used for diagnosis, such as clinical screening, and prognostic evaluation and monitoring of therapy outcome. They can also be used to identify patients most likely to respond to a particular treatment, for drug screening and for drug development. Thus, biomarkers and their use are valuable for identifying new drug therapies and for discovering new targets for drug therapies. Further, they are valuable for exploring dosage regimens and drug combinations.
In the present invention, the "subject" includes all mammals, and a human is selected as one embodiment of the present invention.
The present invention provides a combination of metabolic markers for detecting affective disorders in a subject, comprising dopamine, 5-hydroxytryptamine, gamma-aminobutyric acid, tryptophan, tyramine, kynurenine, 3, 4-dihydroxyphenylacetic acid, epinephrine and norepinephrine, the combination of metabolic markers suitable for the purposes of the present invention may further comprise a combination of one or more of the metabolic markers selected from the group consisting of: 5-hydroxytryptophan, N-acetylserotonin, N-acetylaspartic acid, 5-hydroxyindoleacetic acid, tryptamine, creatine, acetyl-carnitine, creatinine, inositol, phosphorylcholine, adenosine, pyroglutamic acid, tryptol, canine urea, kynurenine, 3-hydroxyaminobenzoic acid, 3-hydroxykynurenine, 3-methoxytryptamine, quinolinic acid, anthranilic acid, alpha-ketoglutaric acid, glutamic acid, ornithine, glutathione, glutamine, succinic acid, phenylalanine, phenylethylamine, tyrosine, valine, leucine, p-hydroxyphenylethylamine, dopa, vanillylmandelic acid, 3-hydroxybutyric acid, homovanillic acid, and choline.
The test sample for detecting the metabolic marker of the present invention may be a blood, urine or other body fluid sample derived from a subject. One embodiment of the present invention is to test urine as a biological sample, and one embodiment is morning urine of a subject. The test body can assist the sample through the professional, or independently take a sample through guiding, including wasing both hands, gathers the midstream urine of morning urine with the urine collection cup, abandons the urine of beginning end, lets the urine of beginning wash the urethra, and it can to leave and get the midspan urine, and the purpose prevents the urine pollution, regards the fresh urine of getting as the detection sample.
The present invention provides a treated urine sample for detecting the above metabolic markers as a detection sample and a method for treating the same; the processing method comprises the steps of attaching the urine sample to a filter paper sheet to prepare a urine filter paper sheet, and drying the urine filter paper sheet to serve as a detection sample. Wherein the urine derived from the subject is preferably morning urine, more preferably midstream urine; the filter paper sheet is selected from quantitative analysis filter paper, qualitative analysis filter paper and slow quantitative ashless filter paper or other filter paper or similar carriers with similar adsorption function.
As a specific embodiment, the preparation of the urine filter paper sheet comprises: (1) cleaning both hands of a collector, preparing to collect articles, and collecting midstream urine of morning urine by using a urine collecting cup; (2) taking out the urine filter paper sheet, keeping the urine filter paper sheet clean, and immersing the urine filter paper sheet into urine for full infiltration; (3) lifting the filter paper sheet, and draining residual urine on the paper surface; (4) and drying the taken filter paper in the dark at 15-30 ℃ in a clean environment. After the filter paper sheet is completely dried, the detection can be directly carried out, or the filter paper sheet can be sealed; as one of the sealing schemes, a piece of filter paper can be put into a sample bag with a sealing strip for sealing; the filter paper sheet should be kept clean during the above process. The urine filter paper sheet sample prepared by the preparation method can be stored for at least 1 year at-80 ℃, can be stored for at least 3 months at-20 ℃ and can be stored for at least 15 days at room temperature. After the sample is taken out from the condition of-80 ℃ or-20 ℃ and is recovered to the normal temperature for detection, the content of the metabolic marker in the sample shows better uniformity and stability.
The metabolic marker combination can be used for preparing a reagent or a kit for detecting the affective disorder of a subject.
The present invention provides a kit comprising a reagent for detecting a metabolic marker as described above, comprising a standard for the metabolic marker, an isotopic internal standard solution, a sodium bicarbonate solution, a derivatizing reagent, and a metabolic marker extraction solvent. The metabolic marker extraction solvent is one or more of water, methanol, ethanol, acetonitrile and isopropanol. The derivatizing agent is selected from dansyl chloride, phenyl isothiocyanate and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.
The kit can be prepared according to the following method:
(1) preparing a standard pure product solution of the metabolic marker;
(2) preparing isotope internal standard solution;
(3) preparing a metabolic marker extraction solvent;
(4) preparing a derivatization reagent;
and assembling the reagents into the kit.
As one scheme of the invention, the kit containing the reagent for detecting the metabolic marker comprises filter paper, a standard substance of the metabolic marker, an isotope internal standard solution, a sodium bicarbonate solution, a derivatization reagent and a metabolic marker extraction agent. The metabolic marker extraction solvent is one or more of water, methanol, ethanol, acetonitrile and isopropanol; the derivatization reagent is selected from dansyl chloride, benzene isothiocyanate and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and the filter paper is selected from analysis filter paper, qualitative analysis filter paper and slow quantitative ashless filter paper.
The use method of the kit comprises the following steps:
(1) preparing a urine filter paper sheet according to the method, wherein the dried urine filter paper sheet is used as a detection sample;
(2) extracting the detection sample dry urine filter paper sheet prepared in the step (1) by using a metabolite extraction solvent and an isotope internal standard solution to obtain an extracting solution;
(3) and (3) determining the extracting solution of the dry urine filter paper sheet by adopting a LC-MS/MS targeted metabonomics analysis method.
The creatinine content in the detection sample is simultaneously measured in the measurement process so as to correct concentration deviation in the sample.
The metabolic markers of the present invention are useful in the diagnosis and treatment of affective disorders in a subject, in the monitoring and assessment of therapeutic drugs, therapeutic efficacy, e.g., clinical screening, and in the prognostic evaluation and monitoring of therapeutic outcome. They can also be used to identify patients most likely to respond to a particular treatment, for drug screening and for drug development; also can be applied to identify new drug treatment and discover new targets of drug treatment. Further, they can be used to explore dosage regimens and drug combinations for the treatment of affective disorders.
Many research institutions have studied diagnostic markers for depression, and no accurate, simple, reliable diagnostic method is widely used clinically. The invention detects the metabolites in the urine filter paper sheet, and can effectively avoid the dependence on the subjective judgment of doctors; the sample is convenient for the patient to collect at home without the cooperation of professional medical staff; the sample is stored on the filter paper sheet, and the filter paper sheet has the advantages of simple and convenient manufacture and collection, convenient transportation, normal-temperature storage and the like. The invention adopts LC-MS/MS method to target and quantify the biomarker to be detected, and the sample detection is accurate and convenient.
Drawings
FIG. 1 is a schematic representation of a urine filter paper sheet made according to the present invention.
FIG. 2 is a liquid chromatography mass spectrometry representative ion chromatogram of a metabolic marker in urine.
FIG. 3 is a graph of principal component analysis scores based on substances in a urine sample.
FIG. 4 is a graph based on orthogonal partial least squares-discriminant analysis (OPLS-DA) of substances detected in urine.
FIG. 5 is a receiver operating characteristic curve (ROC).
Detailed Description
In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a group of metabolic markers, which can be used for diagnosing or assisting in diagnosing affective disorder of a subject. The metabolic markers include dopamine, 5-hydroxytryptamine, gamma-aminobutyric acid, tryptophan, tyramine, kynurenine, 3, 4-dihydroxyphenylacetic acid, epinephrine, norepinephrine, and optionally a combination comprising one or more of the following metabolic markers: 5-hydroxytryptophan, N-acetylserotonin, N-acetylaspartic acid, 5-hydroxyindoleacetic acid, tryptamine, creatine, acetyl-carnitine, creatinine, inositol, phosphorylcholine, adenosine, pyroglutamic acid, tryptol, canine urea, kynurenine, 3-hydroxyaminobenzoic acid, 3-hydroxykynurenine, 3-methoxytryptamine, quinolinic acid, anthranilic acid, alpha-ketoglutaric acid, glutamic acid, ornithine, glutathione, glutamine, succinic acid, phenylalanine, phenylethylamine, tyrosine, valine, leucine, p-hydroxyphenylethylamine, dopa, vanillylmandelic acid, 3-hydroxybutyric acid, homovanillic acid, and choline. The present invention preferably employs a targeted mass spectrometry technique, and other assay techniques such as immunoassay, chemiluminescence, etc. may also be used for the determination of the metabolic markers of the present invention.
The metabolic marker is obtained by screening according to the following method:
(1) selecting depression patients and health control groups (100 cases per group), taking morning urine samples, making into urine filter paper sheet, and extracting metabolite from the urine filter paper sheet;
(2) determining the content of main metabolites in urine by a metabonomics method and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method;
(3) the obtained data is analyzed by orthogonal partial least squares-discriminant analysis (OPLS-DA) and variable projection importance (VIP) to find out metabolites with significant differences between the two groups, and the metabolites are used as alternative metabolic markers;
(4) and further screening and determining a metabolic marker combination with higher accuracy and specificity by a receiver operator characteristic curve (ROC curve).
These metabolic markers may be detected in a urine sample from a subject. The present invention preferably uses urine of a subject as a test sample, particularly morning urine as a test sample, and further preferably, the urine sample may be subjected to the following treatment.
The metabolic markers of the invention can be detected in the form of urine filter paper sheets, i.e. urine samples are attached to filter paper and prepared into the urine filter paper sheets. Filter paper sheets useful in the present invention include, but are not limited to, quantitative analysis filter paper, qualitative analysis filter paper. Preferably, a slow-rate, quantitative ashless filter paper is employed. Carriers having a function similar to filter paper are also suitable for use in the present invention.
The metabolite extraction in the urine filter paper sheet was first performed at the time of measurement. The invention adopts but not limited to water, methanol, ethanol, acetonitrile, isopropanol and the mixed solution of the solvents to extract metabolites in urine, and can contain or not contain isotope internal standards corresponding to each biomarker. Preferably, the metabolites are extracted from the urine filter paper sheet with 5-15% (volume ratio) methanol aqueous solution. Preferably, the extraction is carried out at room temperature (20-35 ℃) with shaking for 20-40 minutes, and the urine filter paper sheet extract is subjected to the subsequent measurement.
In the assay, the present invention preferably employs a derivative method for detecting the metabolic marker. Derivatization methods include, but are not limited to, dansyl chloride, phenyl isothiocyanate, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and the like. Preferably, the derivatization is carried out using dansyl chloride. Preferably, the supernatant of the urine filter paper sheet extract is taken and added with sodium bicarbonate solution and derivatization reagent. Shaking for a period of time to allow sufficient reaction. Preferably, the dansyl chloride derived metabolic marker is extracted with ethyl acetate. The organic phase on the upper layer of the extraction liquid is transferred to a proper container and is dried by nitrogen. Adding methanol water solution for redissolution and detecting.
The invention preferably employs a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect and analyze the diagnostic marker composition. The liquid chromatography comprises High Performance Liquid Chromatography (HPLC), ultra-high performance liquid chromatography (UPLC) and Nano-liter liquid chromatography (Nano-LC), and the tandem mass spectrum comprises Quadrupole mass spectrum (Quadrupole, Q), Time of Flight mass spectrum (Time of Flight, TOF), Ion hydrazine mass spectrum (Ion Trap) and high resolution orbital hydrazine mass spectrum (Orbitrap).
Preferably, the liquid chromatography conditions are: chromatographic column (which can be C8 or C18 silica gel packed column), mobile phase (which can be acetonitrile-water solution, formic acid water solution, methanol-water solution, acetonitrile-isopropanol, methanol-isopropanol solution, acetic acid water solution or ammonium acetate water solution), elution program is: and carrying out gradient elution on the mobile phase A and the mobile phase B according to a certain proportion.
In one embodiment of the invention, the liquid chromatography conditions were such that the liquid chromatography column used BEH C18(1.7 μm, 2.1 x 100mm column). The liquid chromatography conditions were: sample introduction amount: 5 mu L of the solution; flow rate: 0.3 mL/min; column temperature: 40 ℃; autosampler temperature: 10 ℃; mobile phase a phase: 0.1% (volume ratio) formic acid solution; mobile phase B phase: acetonitrile: isopropanol (volume ratio 7: 3); the elution program is 0-1min, and the phase change is from 10% B to 40% B; 1-1.5min, from 40% B phase linear change to 50% B phase; 1.5min-5.5min, linearly changing from 50% B to 100% B phase; 5.5min-7.5min, balancing 100% of phase B; 7.5min-8min, from 100% B phase to 10% B phase; 8min-9min, balance in 10% phase B. Preferably, the detection is performed using triple quadrupole mass spectrometry. Adopting electrospray ionization source (ESI) in positive ion mode, and scanning for 0.5-7 min. The desolventizing gas flow rate is 1000L/h, the taper hole flow rate is 150L/h, the capillary voltage is 3kV, the desolventizing gas temperature is 500 ℃, and the ion source temperature is 150 ℃.
After data acquisition, quantifying the metabolic markers of each diagnostic marker by adopting specific ion pairs, comparing the peak area of the acquired signal with the working curve of the corresponding standard, and correcting by adding an internal standard solution to obtain the concentration values of the metabolic markers. The amount of each metabolic marker in the original urine was corrected for creatinine content.
One embodiment of the present invention is a depression detection kit, comprising: a standard of 5mg/ml metabolic marker,13c stable isotope internal standard solution, 1mg/mL dansyl chloride derivatization reagent solution, sodium bicarbonate solution with the concentration of 1M, methanol-water solution with the volume ratio of 1:9 as a metabolite extraction solvent, and 1 analytical chromatographic column.
Preferably, in the depression detection kit, the metabolic markers include dopamine, 5-hydroxytryptamine, gamma-aminobutyric acid, tryptophan, tyramine, kynurenine, 3, 4-dihydroxyphenylacetic acid, epinephrine and norepinephrine.
The depression detection kit can accurately detect the following metabolic markers in urine: dopamine, 5-hydroxytryptamine, gamma-aminobutyric acid, tryptophan, tyramine, kynurenine, 3, 4-dihydroxyphenylacetic acid, epinephrine and norepinephrine, and is further used for detecting and diagnosing depression and monitoring the drug dosage and the treatment effect in the depression treatment process.
The preparation method of the depression detection kit comprises the following specific preparation steps:
(1) preparing a standard pure product liquid of the metabolic marker: dissolving the metabolite standard substance in water to prepare a solution of 5mg/ml, which is a metabolite standard substance solution;
(2)13preparing a C stable isotope internal standard solution: get13C stable isotope internal standard extract, accurately weighing, dissolving in methanol to prepare 1mg/ml solution, which is13C stable isotope internal standard solution;
(3) preparation of metabolite extraction solvent: mixing analytically pure methanol and water according to the volume ratio of 1:9 to obtain a metabolite extraction solvent; (4) preparation of derivatization reagent: weighing 10mg dansyl chloride solid, dissolving in 10mL acetonitrile to prepare 1mg/mL dansyl chloride acetonitrile solution, preparing as-is-used as far as possible, and storing at-20 ℃ in dark every day.
(5) Analyzing a chromatographic column: as a common chromatography consumable (purchased from Waters corporation, usa), column packing and specifications were: BEH C18(1.7 μm, 2.1 × 100mm column);
the application method of the depression detection kit comprises the following specific steps:
(1) preparing a dry urine filter paper sheet of a subject;
(2) taking methanol-water solution in the depression detection kit as a metabolite extraction solvent and13c, stable isotope internal standard solution, and extracting the metabolite on the dry urine filter paper sheet prepared in the step (1);
(3) and (3) carrying out quantitative determination on 9 metabolites in the dry urine filter paper sheet extracting solution prepared in the step (2) based on an LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) targeted metabonomics analysis method, wherein the metabolites comprise dopamine, 5-hydroxytryptamine, gamma-aminobutyric acid, tryptophan, tyramine, kynurenine, 3, 4-dihydroxyphenylacetic acid, epinephrine and norepinephrine.
Preferably, the application method of the depression detection kit comprises the following specific steps of:
(1) the metabolites in the urine sample were separated by injecting the metabolic extract into a BEH C18 column (2.1 mm. times.100 mm,1.7 μm, Waters, USA) by an autosampler under the following specific chromatographic conditions: sample introduction amount: 5 mu L of the solution; flow rate: 0.3 mL/min; column temperature: 40 ℃; autosampler temperature: 10 ℃; mobile phase a phase: 0.1% (volume ratio) formic acid solution; mobile phase B phase: acetonitrile: isopropanol (volume ratio 7: 3). The above ratios are volume ratios. Elution procedure: phase change from 10% B to 40% B for 0-1 min; 1min-1.5min, linearly changing from 40% B phase to 50% B phase; 1.5min-5.5min, linearly changing from 50% B to 100% B phase; 5.5min-7.5min, balancing 100% of phase B; 7.5min-8min, from 100% B phase to 10% B phase; 8min-9min, balance in 10% phase B.
(2) Introducing the metabolites subjected to chromatographic separation into a triple quadrupole mass spectrum, scanning and detecting the metabolites by adopting a multi-reaction detection mode, and scanning for 0.5-7 min by adopting an electrospray ionization source (ESI) and a positive ion mode. The desolventizing gas flow rate is 1000L/h, the taper hole flow rate is 150L/h, the capillary voltage is 3kV, the desolventizing gas temperature is 500 ℃, and the ion source temperature is 150 ℃.
(3) After data acquisition, quantifying the metabolic markers of each diagnostic marker by adopting specific ion pairs, comparing the peak area of the acquired signal with the working curve of the corresponding standard, and adding an internal standard solution for correction to obtain the concentration values of the metabolic markers. The amount of each metabolic marker in the original urine was corrected for creatinine content.
And (4) carrying out corresponding depression diagnosis risk assessment by comparing the quantitative value of the metabolic marker obtained by the test with the normal value of each metabolite concentration. The concentrations of the 9 metabolites were measured and compared with the normal value of each metabolite concentration, and depression was judged if the concentrations of dopamine, gamma-aminobutyric acid, tyramine, and 3, 4-dihydroxybenzoic acid (greater than 1.5 times the normal value) and tryptophan, kynurenine, 3, 4-hydroxytryptamine, 5-hydroxytryptamine, epinephrine, and norepinephrine were significantly decreased (< 0.8 times the normal value) in the 9 metabolites.
Examples
The technical solution of the present invention is further described with reference to the following specific examples.
Example 1: screening of urine metabolism marker for depression diagnosis
1) Incorporation of the sample
Depression samples inclusion criteria:
1. all included depressed patients met the DSM-IV diagnostic criteria.
2. Hamilton Depression (HDRS)17 item Scale score is greater than or equal to 18.
3. The first-onset depression patients did not take any antidepressant drugs.
4. The age is greater than 18 years and less than 65 years.
Exclusion criteria:
1. those with other mental diseases or previous history of other mental diseases.
2. Combined history of brain organic disease and severe brain trauma; combined with heart, liver and kidney diseases, diabetes and other serious somatic diseases.
3. Abnormal persons are routinely checked in the laboratory (blood routine, liver function, urine routine).
4. Female subjects in pregnancy, lactation, and menstruation.
5. History of drug and substance abuse.
Inclusion and exclusion criteria for healthy controls
No history of neuropsychiatric disease, no history of drug abuse or dependence, no systemic somatic disease; no obvious abnormality is detected in a conventional laboratory.
2) Urine sample is collected to urine filter paper piece
Cutting the filter paper into the shape as shown in figure 1, holding one end of the holding area by hand after collecting fresh urine with a urine cup, vertically immersing the filter paper into the urine, ensuring that the liquid level of the urine infiltrates through a middle straight line, keeping the filter paper to infiltrate vertically for 5 to 10 seconds, and avoiding touching the cup wall as much as possible. And after soaking, vertically taking out the filter paper sheet, slightly putting the front and back surfaces of the filter paper sheet against the cup wall on the upper part without urine, and draining the residual urine on the paper surface. And after collection, placing the urine filter paper in a closed and dry and clean space for drying in the shade, placing the urine filter paper into a sealing bag, and storing the urine filter paper at normal temperature in a dark place or directly using the urine filter paper for sample detection.
3) Biomarker extraction in urine filter paper sheets
(1) Dried urine filter paper samples were punched evenly through 20 holes using an 1/8RD hole punch and collected in a 1.5mL centrifuge tube, with the holes being punched to avoid the edges of the filter paper as much as possible. Adding 500 μ L methanol-water solution (metabolite extraction solvent) in depression detection kit, and oscillating at 20 deg.C and 1450rpm for 45 min;
(2) 10 μ L of the extract was taken and 90 μ L of water was added. Then, 200. mu.L of internal standard working solution, 50. mu.L of 1M NaHCO3 solution and 100. mu.L of 1mg/mL derivatization reagent were added in this order, and the mixture was shaken at 1450rpm for 30 minutes at 30 ℃.
4) Detection of biomarkers in urine filter paper sheets
The LC-MS/MS-based targeted metabonomics analysis method comprises the following specific steps:
(1) introducing the metabolic extract obtained in step 3) into a BEH C18 chromatographic column (2.1mm × 100mm,1.7 μm, Waters, USA) by an autosampler, and separating the metabolites from the urine sample under the following specific chromatographic conditions: sample introduction amount: 5 mu L of the solution; flow rate: 0.3 mL/min; column temperature: 40 ℃; autosampler temperature: 10 ℃; mobile phase a phase: 0.1% formic acid in water; mobile phase B phase: 7:3 acetonitrile: isopropyl alcohol; strong washing liquid: 9:1 methanol: isopropyl alcohol; weak washing liquid: 9:1 acetonitrile: and (3) water. Phase change from 10% B to 40% B for 0-1 min; 1-1.5min, from 40% B phase linear change to 50% B phase; 1.5min-5.5min, linearly changing from 50% B to 100% B phase; 5.5min-7.5min, balancing 100% of phase B; 7.5min-8min, from 100% B phase to 10% B phase; 8min-9min, balance in 10% phase B.
(2) Introducing the metabolites subjected to chromatographic separation into a triple quadrupole mass spectrum, scanning and detecting the metabolites by adopting a multi-reaction detection mode, and scanning for 0.5-7 min by adopting an electrospray ionization source (ESI) and a positive ion mode. The desolventizing gas flow rate is 1000L/h, the taper hole flow rate is 150L/h, the capillary voltage is 3kV, the desolventizing gas temperature is 500 ℃, and the ion source temperature is 150 ℃.
And quantitatively measuring 9 metabolites in the extracting solution of the dry urine filter paper sheet, wherein the 9 metabolites are dopamine, 5-hydroxytryptamine, gamma-aminobutyric acid, tryptophan, tyramine, kynurenine, 3, 4-dihydroxy phenylacetic acid, epinephrine and norepinephrine.
5) Stability test:
after being dried in the air, the urine filter paper is respectively stored at the temperature of minus 80 ℃ and minus 20 ℃ at room temperature. Measuring the content of substances in the urine filter paper sheet by adopting liquid chromatography-mass spectrometry, measuring the sample stored at-80 ℃ once a month, measuring the sample stored at-20 ℃ once a half month, measuring the sample stored at room temperature once every other day, calculating RSD, and calculating the recovery rate by taking the initial measurement mean value as an initial value; the results show that the precision is within 10 percent, the recovery rate is between 80 and 110 percent, and the urine filter paper can be stably stored for at least one year at the temperature of minus 80 ℃, at least 3 at the temperature of minus 20 ℃ and 15 days at room temperature.
6) The result of the detection
200 normal morning urine samples were collected, and the neurotransmitter substances contained in the urine samples were quantified, thereby determining the normal reference range of the markers. The chromatogram of the metabolic marker is shown in FIG. 2. During the urine sample analysis, one QC sample was run through every 10 study samples to assess variability in the derivation process and instrument analysis. QC samples are crucial to ensure reproducibility, reliability, accuracy and robustness (robustness) of quantitative analysis of metabolites. FIG. 3 is a Principal Component Analysis (PCA) score plot. As shown, the QC samples were tightly clustered relative to the other samples of urine, indicating good reproducibility of the method of this example. QC samples here refer to all collected samples including pooled urine of normal and patient urine samples.
Samples of 200 normal persons and 200 depression patients are collected and detected at the same time, and the sample test results show that the normal persons and the depression patients can be well distinguished by the difference of the metabolite levels including neurotransmitter. The metabolites determined included: dopamine, 5-hydroxytryptamine, gamma-aminobutyric acid, tryptophan, tyramine, kynurenine, 3, 4-dihydroxyphenylacetic acid, epinephrine and norepinephrine, 5-hydroxytryptamine, N-acetylpentahydroxytryptamine, N-acetylaspartic acid, 5-hydroxyindoleacetic acid, tryptamine, acetyl-carnitine, creatinine, inositol, phosphorylcholine, adenosine, pyroglutamic acid, tryptol, dog urea, kynurenine, 3-hydroxyaminobenzoic acid, 3-hydroxykynurenine, 3-methoxytryptamine, quinolinic acid, anthranilic acid, alpha-ketoglutaric acid, glutamic acid, ornithine, glutathione, glutamine, succinic acid, phenylalanine, phenylethylamine, tyrosine, valine, leucine, p-hydroxyphenylethylamine, dopa, vanilla mandelic acid, 3-hydroxybutyric acid, valine, tryptophan, and mixtures thereof, Homovanillic acid and choline. Based on the measured urine metabolites, an orthogonal partial least squares-discriminant analysis (OPLS-DA) analysis was performed, as shown in fig. 4, normal and depression samples were well differentiated, indicating that depression patients and normal persons have significant differences in metabolism.
The significantly varying metabolites were further used as receiver operating characteristic curve (ROC), and the area under the curve (AUC) indicated a value of 0.928 for the assay marker for the training set samples, a sensitivity of 93.2%, and a specificity of 96.7%. The validation set data AUC was 0.865, the sensitivity was 83.6%, and the specificity was 89.7%. See fig. 5. The model is proved to have good prediction value. The combination of the metabolites dopamine, 5-hydroxytryptamine, gamma-aminobutyric acid, tryptophan, tyramine, kynurenine, 3, 4-dihydroxyphenylacetic acid, epinephrine, and norepinephrine exhibits excellent specificity and sensitivity.
Example 2: depression detection kit
The depression detection kit comprises: a standard pure solution of the metabolic marker (5mg/ml,1 ml);13c stable isotope internal standard solution (1mg/ml, 100 microliter); metabolite extraction solvent (10 ml); derivatization reagent powder (10mg), analytical column (1). Metabolic markers include: dopamine, 5-hydroxytryptamine, gamma-aminobutyric acid, tryptophan, tyramine, kynurenine, 3, 4-dihydroxyphenylacetic acid, epinephrine, and norepinephrine.
The preparation method of the kit comprises the following steps:
(1) standard pure solutions of metabolic markers: accurately weighing 5mg of metabolite standard substance powder, dissolving in 1ml of water, and preparing into 5mg/ml pure metabolite marker solution;
(2)13c stable isotope internal standard solution: get13C stable isotope extraction powder, accurately weighing 1mg, dissolving in 1ml methanol to obtain 1mg/ml13C stable isotope internal standard solution;
(3) metabolite extraction solvent: accurately measuring 1ml of analytical pure methanol, adding 9ml of chromatographic pure water, and uniformly mixing to obtain a metabolite extract;
(4) analyzing a chromatographic column: is a common chromatographic consumable material, is purchased from Waters company in the United states, and comprises the following chromatographic column packing materials and specifications: BEH C18(1.7 μm, 2.1 × 100mm chromatography column).
The above assembly is a kit.
Example 3: clinical examples of Depression detection kit for diagnosing Depression
Urine from 100 out-patients was collected on a dry urine filter sheet, and the depression was diagnosed using the depression detection kit described in example 2. Extracting metabolite in depression detection kit with solvent (methanol-water solution) and13c, stabilizing isotope internal standard solution, and extracting the metabolite on the dry urine filter paper sheet. The LC-MS/MS-based targeted metabonomics analysis method quantitatively determines 9 metabolites screened in the example 1 in the dry urine filter paper sheet metabolic extract, and comprises the following specific steps:
(1) preparation of urine filter paper sheet
Cutting the filter paper into the shape as shown in figure 1, holding one end of the holding area by hand after collecting fresh urine with a urine cup, vertically immersing the filter paper into the urine, ensuring that the liquid level of the urine infiltrates through a middle straight line, keeping the filter paper to infiltrate vertically for 5 to 10 seconds, and avoiding touching the cup wall as much as possible. And after soaking, vertically taking out the filter paper sheet, slightly putting the front and back surfaces of the filter paper sheet against the cup wall on the upper part without urine, and draining the residual urine on the paper surface. And after collection, placing the urine filter paper sheets in a closed and dry and clean space for drying in the shade, placing the urine filter paper sheets into a sealing bag, and storing the urine filter paper sheets at normal temperature in a dark place or directly using the urine filter paper sheets for detection.
(2) Extraction of metabolic markers from urine filter paper
Dried urine filter paper samples were punched evenly through 20 holes using an 1/8RD hole punch and collected in a 1.5mL centrifuge tube, with the holes being punched to avoid the edges of the filter paper as much as possible. Adding 500 μ L methanol-water solution (metabolite extraction solvent) in depression detection kit, and oscillating at 20 deg.C and 1450rpm for 45 min; obtaining the urine filter paper extracting solution.
(3) Derivatization treatment
Taking 10 mu L of the urine filter paper sheet extract obtained in the step (2), and adding 90 microliter of water. Then, 200 microliters of internal standard solution, 50 microliters of 1M NaHCO3 solution and 100 microliters of derivatization reagent are sequentially added, and the reaction is carried out at 30 ℃ and 1450rpm for 30 minutes; to the reacted tube was added 500. mu.l of ethyl acetate, and the mixture was shaken at 1450rpm for 20 minutes at 20 ℃. 400 microliters of the upper organic phase was transferred to a 1mL 96-well plate and blown dry with nitrogen. 100 microliters of 50% methanol aqueous solution was added for reconstitution, and the mixture was shaken at 650rpm for 20 minutes at 10 ℃. Centrifuging, and taking supernatant for detection.
(4) Determination of metabolites in a sample
The supernatant obtained in step (3) was injected into a BEH C18 column (2.1 mm. times.100 mm,1.7 μm, Waters, USA) by means of an autosampler to separate the metabolites from the urine sample, under the following specific chromatographic conditions: sample introduction amount: 5 mu L of the solution; flow rate: 0.3 mL/min; column temperature: 40 ℃; autosampler temperature: 10 ℃; mobile phase a phase: 0.1% formic acid in water; mobile phase B phase: 7:3 acetonitrile: isopropyl alcohol; strong washing liquid: 9:1 methanol: isopropyl alcohol; weak washing liquid: 9:1 acetonitrile: and (3) water. Phase change from 10% B to 40% B for 0-1 min; 1-1.5min, from 40% B phase linear change to 50% B phase; 1.5min-5.5min, linearly changing from 50% B to 100% B phase; 5.5min-7.5min, balancing 100% of phase B; 7.5min-8min, from 100% B phase to 10% B phase; 8min-9min, balance in 10% phase B.
Introducing the metabolites subjected to chromatographic separation into a triple quadrupole mass spectrum, scanning and detecting the metabolites by adopting a multi-reaction detection mode, and scanning for 0.5-7 min by adopting an electrospray ionization source (ESI) and a positive ion mode. The desolventizing gas flow rate is 1000L/h, the taper hole flow rate is 150L/h, the capillary voltage is 3kV, the desolventizing gas temperature is 500 ℃, and the ion source temperature is 150 ℃.
The concentrations of the 9 metabolites were compared with the normal metabolite concentration, and depression was judged if the concentrations of dopamine, gamma-aminobutyric acid, tyramine and 3, 4-dihydroxybenzoic acid were significantly increased (1.5 times greater than the normal value) and the concentrations of tryptophan, kynurenine, 3, 4-dihydroxybenzoic acid, 5-hydroxytryptamine, epinephrine and norepinephrine were significantly decreased (< 0.8 times the normal value) in the 9 metabolites.
At the same time as this experiment was performed, depression was diagnosed by a medical professional on 100 persons who had a medical visit using the questionnaire, and the results are shown in table 1 as the gold standard for diagnosis compared with the kit test method provided by the present invention. The accuracy of the metabolomic marker kit diagnosis of depression from table 1 reached 93.0% ((47+46)/100) × 100%, specificity 92.0% (46/(46 +4)) × 100%, and sensitivity 94.0% (47/(47+3)) × 100%.
TABLE 1 evaluation of efficacy of metabolic marker kit for diagnosing depression
Example 4: depression detection kit
The depression detection kit comprises: a standard pure solution of the metabolic marker (5mg/ml,1 ml);13c stable isotope internal standard solution (1mg/ml, 100 microliter); metabolite extraction solvent (10 ml); derivatization reagent powder (10mg), analytical chromatography column (1), and filter paper. The metabolic markers include: dopamine, 5-hydroxytryptamine, gamma-aminobutyric acid, tryptophan, tyramine, kynurenine, 3, 4-dihydroxyphenylacetic acid, epinephrine, and norepinephrine. The filter paper is in a sealed state.
The preparation method of the kit comprises the following steps:
(1) standard pure solutions of metabolic markers: accurately weighing 5mg of metabolite standard substance powder, dissolving in 1ml of water, and preparing into 5mg/ml pure metabolite marker solution;
(2)13c stable isotope internal standard solution: get13C stable isotope extraction powder, accurately weighing 1mg, dissolving in 1ml methanol to obtain 1mg/ml13C stable isotope internal standard solution;
(3) metabolite extraction solvent: accurately measuring 1ml of analytical pure methanol, adding 9ml of chromatographic pure water, and uniformly mixing to obtain a metabolite extract;
(4) analyzing a chromatographic column: is a common chromatographic consumable material, is purchased from Waters company in the United states, and comprises the following chromatographic column packing materials and specifications: BEH C18(1.7 μm, 2.1 × 100mm column);
(5) filter paper: cutting ashless filter paper into the shape shown in figure 1, and sealing with a sealing bag.
The above assembly is a kit.
The above detailed description of the use of the urine metabolism marker and the depression detecting kit with reference to the examples is illustrative and not restrictive, and several examples may be cited within the scope of the present invention, so that variations and modifications without departing from the general concept of the present invention shall fall within the scope of the present invention.
Claims (11)
1. Metabolic markers for detecting affective disorders in a subject, including dopamine, 5-hydroxytryptamine, gamma-aminobutyric acid, tryptophan, tyramine, kynurenine, 3, 4-dihydroxybenzeneacetic acid, epinephrine, and norepinephrine; the affective disorder is selected from depression, schizophrenia and anxiety, and the depression is selected from dysthymia, endogenous depression, reactive depression, mild depression, major depression, psychotic depression, nervous depression, unipolar depression and bipolar depression.
2. The metabolic marker for detecting affective disorders in a subject of claim 1, further comprising a combination of one or more of the following metabolic markers: 5-hydroxytryptophan, N-acetylserotonin, N-acetylaspartic acid, 5-hydroxyindoleacetic acid, tryptamine, creatine, acetyl-carnitine, creatinine, inositol, phosphorylcholine, adenosine, pyroglutamic acid, tryptol, canine urea, kynurenine, 3-hydroxyaminobenzoic acid, 3-hydroxykynurenine, 3-methoxytryptamine, quinolinic acid, anthranilic acid, alpha-ketoglutaric acid, glutamic acid, ornithine, glutathione, glutamine, succinic acid, phenylalanine, phenylethylamine, tyrosine, valine, leucine, p-hydroxyphenylethylamine, dopa, vanillylmandelic acid, 3-hydroxybutyric acid, homovanillic acid, and choline.
3. The test sample for detecting the metabolic marker according to any one of claims 1 or 2, which is obtained by a method comprising obtaining a biological sample of the test sample from urine of a subject; the processing method comprises the steps of attaching urine to a filter paper sheet to prepare a urine filter paper sheet, and drying the urine filter paper sheet to serve as a detection sample.
4. The test sample according to claim 3, wherein the urine of the subject is morning urine.
5. The test sample of claim 3, wherein the filter paper sheet is selected from the group consisting of analytical filter paper, qualitative analytical filter paper, and slow-rate quantitative ashless filter paper.
6. Use of the metabolic marker of any one of claims 1 or 2 for the preparation of a reagent for detecting an affective disorder selected from the group consisting of depression, schizophrenia and anxiety in a subject selected from the group consisting of dysthymia, endogenous depression, reactive depression, mild depression, major depression, psychotic depression, neurological depression, unipolar depression and bipolar depression.
7. A kit comprising reagents for detecting the metabolic marker of any of claims 1 or 2, comprising a standard for the metabolic marker, an isotopic internal standard solution, a sodium bicarbonate solution, a derivatizing reagent, and a metabolic marker extracting agent.
8. The kit according to claim 7, wherein the metabolic marker extracting solvent is selected from one or more of water, methanol, ethanol, acetonitrile and isopropanol; the derivatizing agent is selected from dansyl chloride, phenyl isothiocyanate and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.
9. A kit comprising reagents for detecting the metabolic marker of any of claims 1 or 2, comprising filter paper, a standard for the metabolic marker, an isotopic internal standard solution, a sodium bicarbonate solution, a derivatizing reagent, and a metabolic marker extracting agent.
10. The kit according to claim 9, wherein the metabolic marker extracting solvent is selected from one or more of water, methanol, ethanol, acetonitrile and isopropanol; the derivatization reagent is selected from dansyl chloride, benzene isothiocyanate and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and the filter paper is selected from analysis filter paper, qualitative analysis filter paper and slow quantitative ashless filter paper.
11. A method of using the kit of any one of claims 9 to 10, comprising:
(1) the method for treating a test sample according to any one of claims 3 to 5, wherein a piece of urine filter paper is prepared, and the dried piece of urine filter paper is used as the test sample;
(2) extracting the detection sample dry urine filter paper sheet prepared in the step (1) by using a metabolite extraction solvent and an isotope internal standard solution to obtain an extracting solution;
(3) the metabolic markers were measured on dry urine filter paper extract using LC-MS/MS targeted metabonomics analysis with creatinine correction.
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