CN113552228A - Combined markers for diagnosing childhood bronchiolitis and application and detection kit thereof - Google Patents
Combined markers for diagnosing childhood bronchiolitis and application and detection kit thereof Download PDFInfo
- Publication number
- CN113552228A CN113552228A CN202010331763.XA CN202010331763A CN113552228A CN 113552228 A CN113552228 A CN 113552228A CN 202010331763 A CN202010331763 A CN 202010331763A CN 113552228 A CN113552228 A CN 113552228A
- Authority
- CN
- China
- Prior art keywords
- bronchiolitis
- tryptophan
- subject
- kit
- serum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 206010006448 Bronchiolitis Diseases 0.000 title claims abstract description 55
- 238000001514 detection method Methods 0.000 title abstract description 11
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims abstract description 36
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims abstract description 36
- 210000002966 serum Anatomy 0.000 claims abstract description 35
- 208000006673 asthma Diseases 0.000 claims abstract description 32
- GKZIWHRNKRBEOH-UHFFFAOYSA-N phenylalanylphenylalanine Chemical compound C=1C=CC=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 GKZIWHRNKRBEOH-UHFFFAOYSA-N 0.000 claims abstract description 30
- 108010073025 phenylalanylphenylalanine Proteins 0.000 claims abstract description 30
- 239000002207 metabolite Substances 0.000 claims abstract description 26
- 239000003550 marker Substances 0.000 claims abstract description 19
- 238000007477 logistic regression Methods 0.000 claims abstract description 6
- 150000003384 small molecules Chemical class 0.000 claims abstract description 4
- 239000000126 substance Substances 0.000 claims description 14
- 150000002500 ions Chemical class 0.000 claims description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 12
- 238000004458 analytical method Methods 0.000 claims description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 6
- 235000019253 formic acid Nutrition 0.000 claims description 6
- 230000002503 metabolic effect Effects 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 238000012937 correction Methods 0.000 claims description 3
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 2
- 230000014759 maintenance of location Effects 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims 2
- 239000007788 liquid Substances 0.000 claims 1
- 238000004451 qualitative analysis Methods 0.000 claims 1
- 206010006451 bronchitis Diseases 0.000 abstract description 18
- 238000003745 diagnosis Methods 0.000 abstract description 13
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 238000011161 development Methods 0.000 abstract description 5
- 238000003759 clinical diagnosis Methods 0.000 abstract description 3
- 239000000523 sample Substances 0.000 description 17
- 238000000034 method Methods 0.000 description 8
- 239000003643 water by type Substances 0.000 description 7
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 6
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000010200 validation analysis Methods 0.000 description 4
- 201000009961 allergic asthma Diseases 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000037149 energy metabolism Effects 0.000 description 3
- 210000003296 saliva Anatomy 0.000 description 3
- 208000000059 Dyspnea Diseases 0.000 description 2
- 206010013975 Dyspnoeas Diseases 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 108010058846 Ovalbumin Proteins 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 150000002066 eicosanoids Chemical class 0.000 description 2
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 2
- 230000004199 lung function Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 229940092253 ovalbumin Drugs 0.000 description 2
- 230000006920 protein precipitation Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000026618 small molecule metabolic process Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 2
- UCTWMZQNUQWSLP-VIFPVBQESA-N (R)-adrenaline Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-N 0.000 description 1
- 229930182837 (R)-adrenaline Natural products 0.000 description 1
- PHIQHXFUZVPYII-ZCFIWIBFSA-O (R)-carnitinium Chemical compound C[N+](C)(C)C[C@H](O)CC(O)=O PHIQHXFUZVPYII-ZCFIWIBFSA-O 0.000 description 1
- 206010003504 Aspiration Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 description 1
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 239000000956 alloy Substances 0.000 description 1
- 229910045601 alloy Inorganic materials 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960004203 carnitine Drugs 0.000 description 1
- 238000011976 chest X-ray Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 229960005139 epinephrine Drugs 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- 208000015707 frontal fibrosing alopecia Diseases 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 208000030603 inherited susceptibility to asthma Diseases 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000003871 intestinal function Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 238000002705 metabolomic analysis Methods 0.000 description 1
- 230000001431 metabolomic effect Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000035778 pathophysiological process Effects 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 230000004202 respiratory function Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940034208 thyroxine Drugs 0.000 description 1
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- 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
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
-
- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
Landscapes
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to a new application of small molecule metabolites tryptophan and phenylalanyl phenylalanine in a serum sample as a combined marker in preparing a kit for diagnosing children bronchiolitis. The present invention also relates to a kit for detecting bronchiolitis in a subject, which judges whether the subject has bronchiolitis by detecting the respective concentrations of the above-mentioned combination markers in serum from the subject, calculating the combination marker variables Prob and a judgment cut-off value based on a binary logistic regression equation. The kit has the characteristics of low detection cost and good stability. The invention can be applied to the clinical diagnosis of the auxiliary capillary bronchitis, can effectively distinguish the capillary bronchitis from the asthma, has the characteristics of high diagnosis specificity and high sensitivity, and has higher development and application values.
Description
Technical Field
The present invention relates to the fields of analytical chemistry and clinical medicine. In particular, the invention relates to a kit for distinguishing bronchiolitis by taking tryptophan and phenylalanyl phenylalanine as combined markers.
Background
Bronchiolitis is a common acute lower respiratory infection in children, and occurs mostly in infants under 2 years of age, especially in infants less than 6 months. Bronchiolitis and Asthma are closely related, both with typical asthmatic symptoms, and some are actually the first episodes of Asthma (document 1: Ma Y, Hospital T B. research Progress on the Relationship of the Relationship Between Between brochialities and assay [ J ]. Heilongjiang Medicine Journal, 2016). The prevalence of asthma (22.1% -53.2%) after illness in children with bronchiolitis is much higher than that of natural asthma (0.25% -4.63%) in children in China (document 2: Johnson C H, Ivanisevic J, Siuzdak G. metals: bearings and dynamics mechanisms [ J ]. Nature Reviews Molecular Biology, Cell Biology,2016,17(7): 451) 459). But there is currently a lack of clinically effective methods for distinguishing between asthma and bronchiolitis. In addition, bronchitis is also a common lower respiratory disease. In terms of clinical manifestations, bronchitis does not cause dyspnea and does not cause breathlessness. When the lung function is detected, the lung function of a patient with capillary bronchitis is accompanied by functional obstruction; patients with bronchitis are not accompanied by obstruction of the respiratory function of the respiratory tract. Therefore, the two can be distinguished more clearly. Currently, the clinical diagnosis of infantile bronchiolitis is mainly based on percutaneous blood oxygen saturation monitoring, nasopharyngeal aspiration etiology detection, chest X-ray examination, and the like. All three have universal applicability, and the diagnosis is complex and the cost is high. Therefore, development of a novel diagnostic method with high sensitivity and specificity is imperative.
Metabolomics combines high-throughput analysis techniques with bioinformatics to perform comprehensive analysis of metabolites in biological samples. In recent years, breath condensate (EBC) and alveolar lavage are the most common samples used in studies on bronchiolitis. Cruickshank-Quinn et al found that the content of eicosanoids in saliva was several orders of magnitude higher than in EBCs without saliva contamination, so the concentration of eicosanoids in EBCs reported previously could be due to saliva contamination (reference 3: Cruickshank-Quinn C, Armstrong M, Powell R, et al. Although changes in metabolites in alveolar lavage fluid reflect more intuitively the pathogenesis of bronchial asthma, it is invasive and limited in human studies, and Quinn et al demonstrate that metabolites in serum can be a replacement for pulmonary metabolites (document 4: Quinn K D, Schedel M, Nkrumah-Elie Y, et al. Dysregistration of metabolic pathways in a motor model of allergic asthma [ J ]. Allergy,2017,72 (9)). Therefore, it is very important to detect the content of metabolites in the serum of patients with asthma and bronchiolitis by using an efficient and high-throughput metabonomics technology. The biomarker of the bronchiolitis is determined by a metabonomics technology, the severity of the disease can be predicted and layered, and the method has important significance for clinical prediction and treatment.
The invention utilizes ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) technology to detect the serum metabolic profiles of patients with capillary bronchitis, asthmatic patients and normal population, divides the serum metabolic profiles into a group with capillary bronchitis and a group with non-capillary bronchitis (including normal population and asthmatic patients with interference disease), optimizes a combined marker of tryptophan and phenylalanyl phenylalanine through binary logistic regression analysis, and is used for diagnosing the capillary bronchitis in a subject at one time. Tryptophan and phenylalanyl phenylalanine are involved in various pathophysiological processes in the human body. Tryptophan is an important amino acid and an important energy metabolism precursor. Studies have reported that the decrease in tryptophan content in the plasma of ovalbumin-induced mice suggests that energy metabolism plays an abnormal role in the development of ovalbumin-induced allergic asthma (reference 5: Abserant pure metabolism in allergic asthma by plasma metabolism [ J ]. Journal of Pharmaceutical & biological Analysis 2016,120: 181-189). Phenylalanine can be converted into tyrosine, and the simultaneous increase of both promotes the synthesis of epinephrine and thyroxine, further regulating energy metabolism (document 6: Hsu J W, Ball R O, Pencharz P B. the existence of the polypeptide which is a phenylalkamine may not provide for the production of amino acids in the chittree [ J ]. Pediatric Research,2007,61(3): 361.). In addition, phenylalanine and tyrosine have the function of regulating intestinal functions. Previous studies have confirmed that the imbalance of intestinal flora is closely related to the occurrence and development of immune disorders and variable diseases of the body, such as asthma, bronchiolitis, etc. (document 7: Mcloughlin R M, Mills K H G. infection of organic structural bacteria on the immune responses at medium alloy and asthma [ J ]. Journal of organic & Clinical Immunology,2011,127(5): 1097. sup. 1107). At present, no report exists for using the combined marker in diagnosis of bronchiolitis.
Disclosure of Invention
The invention aims to solve the problem of difficult diagnosis of the bronchiolitis, provides application of a combined small molecule metabolite in auxiliary diagnosis of the bronchiolitis, and provides an analysis detection method of the combined small molecule metabolite.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
(1) metabonomic fingerprint analysis is carried out on serum of patients with capillary bronchitis, asthma and normal human by using a high performance liquid chromatography-mass spectrometry combined metabonomic technology;
(2) carrying out nonparametric inspection on quantifiable metabolites by using MEV software, and calculating false positive rate (FDR) values and p values of all the metabolites, wherein the metabolites with the FDR values of <0.05 and the p values of <0.05 have significant difference, so that the significant difference exists between the 37 metabolites in non-bronchiolitis (including normal population and patients suffering from the interference asthma) and patients suffering from the bronchiolitis, wherein the metabolites comprise carnitine, Sphingomyelin (SMs), Phosphatidylethanolamine (PEs), fatty acids (FFAs), amino acids and the like (the 37 different metabolites and the relative content thereof are shown in the attached table);
(3) using the data statistics software SPSS, by the binary logistic regression analysis method, by forward: the condition method screens 37 different metabolites, screens five groups of markers, and then evaluates the sensitivity and specificity of the screened combined markers and the area under the curve (AUC) by using an ROC (receiver operating characterization) curve (shown in Table 1). The specificity and the sensitivity are both high, and the combination which simultaneously gives consideration to the simplicity (namely the number of metabolites participating in the combination is less) can be used as a combined marker, and tryptophan and phenylalanyl phenylalanine are selected as the combined marker for auxiliary diagnosis of the capillary bronchitis;
TABLE 1 evaluation results of combination markers
(4) Verifying the combined metabolic marker by applying another batch of serum samples of patients with capillary bronchitis, patients with asthma and normal human, and determining that tryptophan and phenylalanyl phenylalanine can be used as combined markers for auxiliary diagnosis of capillary bronchitis;
(5) use of combination markers: the serum concentrations of tryptophan and phenylalanine are reduced in bronchiolitis patients relative to non-bronchiolitis patients (normal population and asthmatic patients). Using data statistics software SPSS to regress the metabolites into a combined marker variable P by a binary logistic regression method, the equation of which is as follows:
P＝1/(1+e-(-8.375·a-15.964+b+6.959))
wherein a is the concentration of tryptophan in the serum sample, and b is the concentration of phenylalanyl phenylalanine in the serum sample. The variable P is increased in patients with bronchiolitis, and the variable value can be used for assisting in judging the bronchiolitis. The cut-off value of the combined marker determined by the invention for judging the bronchiolitis is set to be 0.365, and the bronchiolitis is possible if the cut-off value is higher than the cut-off value.
(6) The diagnostic system comprises means for: the column was a Waters BEH C8 column (100 mm. times.2.1 mm,1.7 μm) (Waters, Milford, Mass.), the separation system was Waters ACQUITY UPLC, the detection system was Triple TOF mass spectrometry, using positive ion mode detection;
(7) determining the optimal composition of the kit:
a. and (3) standard substance: tryptophan and phenylalanyl phenylalanine. The standards were used to characterize the corresponding serum metabolites tryptophan and phenylalanyl phenylalanine, respectively. Performing liquid chromatography-mass spectrometry on two substance standards with the concentrations of 1-10 mug/mL, determining the chromatographic retention time of the two standards and the actually measured mass-to-charge ratio of the two ions, and comparing the two substances with the two substances actually measured in a sample of a subject;
b. extract used for serum sample pretreatment: the extract was used to pre-condition serum samples from subjects as a methanol solution containing 4.25 μ g/mL of D5-tryptophan as an internal standard. D5-Tryptophan was used to correct for Tryptophan and phenylalanyl phenylalanine. Respectively comparing the ion peak intensities of the two qualitative substances in each sample of the tested person with the internal standard substance in the extracting solution, and obtaining the relative concentration of tryptophan and phenylalanyl phenylalanine by internal standard correction;
c. eluent: 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile; the invention has the following effects:
the combined marker variable P in serum can be used for well diagnosing the bronchiolitis. The detection kit provided by the invention has the advantages of simplicity, convenience, rapidness and good repeatability in the detection of the metabolite combination, and is suitable for assisting the clinical diagnosis of the capillary bronchitis. The sensitivity and specificity and area under the curve (AUC) are given in table 2 below.
TABLE 2 results of the use of combination markers
Drawings
FIG. 1. the relative amounts of tryptophan and phenylalanine in the discovery and validation sets were varied in each group of serum samples (mean + standard deviation).
Fig. 2 (a) ROC plot of combined markers in the discovery set for diagnosis of bronchiolitis, AUC 0.967; (B) the combination markers were used in the validation set to diagnose the ROC profile of bronchiolitis, AUC 1.
Detailed Description
Example 1
1. Serum sample collection
All volunteers enrolled in the study signed an informed consent prior to serum sample collection. Blood samples were collected from 20 bronchiolitis patients (1 month-19 months), 22 asthma patients (0.5-13 years) and 26 healthy persons (0-6 years) under the same conditions, and after collecting and standing for 60 minutes, serum was directly collected and stored in a refrigerator at-80 ℃ for further use.
2. Analytical method
2.1 serum sample pretreatment
To a 96-well protein precipitation plate was added 200. mu.L of methanol containing internal standard (containing 1 internal standard: 4.25. mu.g/mL D5-tryptophan), 50. mu.L of serum was added, vortexed at low speed for 10min, and then centrifuged at 400g, the upper protein precipitation plate was discarded, and the sample in the lower collection cassette was lyophilized. Before LC-MS analysis, the sample in the collection box was redissolved with 80. mu.L acetonitrile/water at a volume ratio of 1/4, vortexed for 10min, centrifuged to take the supernatant, transferred into a sample bottle, and analyzed by sample injection, with a sample amount of 5. mu.L.
2.2 apparatus conditions
The liquid chromatography system used was a Waters acquisition UPLC (Waters Corp, Milford, USA). A chromatographic column: waters BEH C8 column (100mm × 2.1mm,1.7 μm) (Waters, Milford, MA), column temperature: 50 ℃, flow rate: 0.35 ml/min. Mobile phase: water added 0.1% formic acid (phase a) and acetonitrile added 0.1% formic acid (phase B). Gradient: the initial gradient was 10% B for 1min, followed by a linear increase to 40% B within 4min, a further linear increase to 100% B within 12min and a 5min hold, a drop back to the initial gradient of 10% B at 22.1min, and an equilibration time of 2.9 min.
The detection system is Triple TOFTM5600+ mass spectrum (AB SCIEX, Framingham, USA), positive ion mode. TOF full scan range m/z 50-1200; curtain Gas 0.241MPa, GS10.276 MPa, GS20.276 MPa; the declustering voltage of the compound is 100V; collision energy is 10V; IDA-based auto-MS2 (automatic secondary fragment scan selected to respond to the first 20 ions higher for auxiliary characterization) m/z range: 50-1200; the collision energy was 30V, and the collision energy extended range 10.
3. Serum test result and auxiliary diagnosis method
Extracting peak areas of the combined markers tryptophan, phenylalanyl phenylalanine and an internal standard compound, carrying out internal standard correction on the peak areas of the metabolites to obtain corresponding relative concentrations, and correcting the tryptophan and the phenylalanyl phenylalanine by adopting D5-tryptophan. Tryptophan and phenylalanyl phenylalanine were quantitatively analyzed. The relative amounts of the above metabolites in the normal control group, bronchiolitis group and asthma group are shown in fig. 1 (finding set) and table 3.
TABLE 3 relative amounts of tryptophan and phenylalanyl phenylalanine in Normal controls, asthma and bronchiolitis
The serum concentrations of tryptophan and phenylalanine are reduced in bronchiolitis patients relative to non-bronchiolitis patients (normal population and asthmatic patients). Meanwhile, substituting the relative content of each metabolite into SPSS software to perform binary logic modeling analysis, wherein the regression equation of the built model is as follows:
P＝1/(1+e-(-8.375·a-15.964+b+6.959))
wherein a is the concentration of tryptophan in the serum sample, and b is the concentration of phenylalanyl phenylalanine in the serum sample. The variable P is increased in patients with bronchiolitis, and the variable value can be used for assisting in judging the bronchiolitis. The cut-off value of the combined marker determined by the invention for judging the bronchiolitis is set to be 0.365, and the bronchiolitis is possible if the cut-off value is higher than the cut-off value.
And (3) making an ROC curve for diagnosing the capillary bronchitis by using the tryptophan and phenylalanyl phenylalanine combination marker, and evaluating the applicability of the combination marker to the discrimination of the capillary bronchitis. The small molecule metabolism combination marker has better discrimination capability and better diagnosis effect on non-bronchiolitis (normal control and asthma) and bronchiolitis. AUC was 0.967, sensitivity was 100%, and specificity was 91.7% (see table 4 and fig. 2).
Table 4.
Example 2
1. Serum sample collection
All volunteers enrolled in the study signed an informed consent prior to serum sample collection. Blood samples were collected from 10 bronchiolitis patients (1 month-19 months), 11 asthma patients (0.5-13 years) and 12 healthy persons (0-6 years) under the same conditions, and after collecting and standing for 60 minutes, serum was directly collected and stored in a refrigerator at-80 ℃ for further use.
2. Analytical method
Same as example 1
3. Serum test result and auxiliary diagnosis method
Example 2 the results of the validation set substantially matched the results of the discovery set of example 1. The relative amounts of tryptophan and phenylalanine in the normal control group, bronchiolitis group and asthma group are shown in fig. 1 (validation set) and table 5. The serum concentrations of tryptophan and phenylalanylphenylalanine were reduced in bronchiolitis patients relative to the normal control and asthma groups.
TABLE 5 relative amounts of tryptophan and phenylalanyl phenylalanine in Normal controls, asthma and bronchiolitis
The relative concentrations of the metabolites were respectively substituted into the binary logistic regression equation obtained in example 1 and the cutoff values obtained in example 1 were used to determine the diagnostic effect. The small molecule metabolism combination marker has better discrimination capability and better diagnosis effect on non-bronchiolitis (normal control and asthma) and bronchiolitis. AUC was 1, sensitivity was 100.0%, and specificity was 91.3% (see table 6 and fig. 2).
Table 6.
The kit has the characteristics of low detection cost and good stability. Meanwhile, the invention can effectively distinguish the capillary bronchitis and the asthma, has the characteristics of high diagnosis specificity and high sensitivity, and has higher development and application values.
It should be understood that while the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein, and any combination of the various embodiments may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.
Appendix 37 different metabolites and their relative contents
Claims (10)
1. A group of combined markers for diagnosing children bronchiolitis consists of combined metabolic markers tryptophan and phenylalanyl phenylalanine.
2. Use of a combination marker for the manufacture of a kit for serodiagnosis of a patient with bronchiolitis in a subject, said combination metabolic marker comprising: tryptophan and phenylalanylphenylalanine, or consisting of tryptophan and phenylalanylphenylalanine.
3. A kit for detecting bronchiolitis in a subject, comprising: tryptophan and phenylalanyl phenylalanine, said subject being a child under the age of 14 years.
4. The kit of claim 3, comprising:
(1) and (3) standard substance: the standard substances are respectively used for the qualitative determination of metabolites tryptophan and phenylalanylphenylalanine in corresponding serum, and the concentration is 1-10 mug/mL;
(2) extracting solution containing internal standard: the extract is used for pre-treating a serum sample from a subject as a methanol solution comprising 4-4.5 μ g/mL, preferably 4.25 μ g/mL of D5-tryptophan as an internal standard;
(3) eluent: 0.05-0.2%, preferably 0.1% (v/v) formic acid in water, 0.05-0.2%, preferably 0.1% (v/v) formic acid in acetonitrile.
5. The kit according to claim 4, wherein the subject is a person to be examined for bronchiolitis including bronchiolitis patients, asthma patients and normal persons, preferably the subject is a child under 14 years of age.
6. The kit of claim 4, wherein the ion peak intensities of tryptophan and phenylalanyl phenylalanine are extracted from the total ion flow graph obtained after the serum sample of the subject is detected by a liquid chromatography-mass spectrometer; the extraction parameters of tryptophan were: in positive ion mode, the mass-to-charge ratio is 205.0972 +/-0.005, and the extraction parameters of phenylalanyl phenylalanine are as follows: positive ion mode, mass to charge ratio 313.1547 ± 0.005.
7. The kit according to claim 4, wherein the two substances tryptophan and phenylalanylphenylalanine standard in the kit characterize the detected ions; two substance standards at concentrations of 1-10 μ g/mL were subjected to liquid chromatography-mass spectrometry analysis to determine chromatographic retention times for the two standards and the measured mass-to-charge ratios of the two ions, compared to the two substances measured in the subject sample.
8. The kit according to claim 6 or 7, wherein the two substances tryptophan and phenylalanylphenylalanine standard is used for quantifying the detected ions, and the ion peak intensities of the two substances after qualitative analysis in each sample of the subject are respectively compared with the internal standard substance in the extracting solution, and the relative concentrations of tryptophan and phenylalanylphenylalanine are obtained through the internal standard D5-tryptophan correction.
9. The kit according to claim 8, wherein the combined marker variable Prob value and the judgment intercept value are calculated by a binary logistic regression equation based on the relative concentration values of the two small molecule metabolites;
the regression equation for the model built is as follows:
P＝1/(1+e-(-8.385*a-15.964*b+6.959))
wherein, a is the concentration of tryptophan in the serum sample, and b is the concentration of phenylalanyl phenylalanine in the serum sample; the variable P is increased in patients with bronchiolitis, and the variable value can be used for assisting in judging the bronchiolitis.
10. The kit according to claim 9, wherein the determined cutoff value for the determination of bronchiolitis by the combination marker is set to 0.365, and bronchiolitis is possible if the cutoff value is higher than the determined cutoff value, and not otherwise.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010331763.XA CN113552228A (en) | 2020-04-24 | 2020-04-24 | Combined markers for diagnosing childhood bronchiolitis and application and detection kit thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010331763.XA CN113552228A (en) | 2020-04-24 | 2020-04-24 | Combined markers for diagnosing childhood bronchiolitis and application and detection kit thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113552228A true CN113552228A (en) | 2021-10-26 |
Family
ID=78101232
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010331763.XA Pending CN113552228A (en) | 2020-04-24 | 2020-04-24 | Combined markers for diagnosing childhood bronchiolitis and application and detection kit thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113552228A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114544812A (en) * | 2022-02-18 | 2022-05-27 | 复旦大学附属中山医院 | Application of metabolic combination type marker in asthma diagnosis |
-
2020
- 2020-04-24 CN CN202010331763.XA patent/CN113552228A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114544812A (en) * | 2022-02-18 | 2022-05-27 | 复旦大学附属中山医院 | Application of metabolic combination type marker in asthma diagnosis |
| CN114544812B (en) * | 2022-02-18 | 2023-06-30 | 复旦大学附属中山医院 | Application of metabolic combination type marker in diagnosis of asthma |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2016204969B2 (en) | Metabolic biomarkers of autism | |
| CN111289736A (en) | Slow obstructive pulmonary early diagnosis marker based on metabonomics and application thereof | |
| Maniscalco et al. | Clinical metabolomics of exhaled breath condensate in chronic respiratory diseases | |
| US20150090010A1 (en) | Method for diagnosing heart failure | |
| Liang et al. | Metabolomics of alcoholic liver disease: a clinical discovery study | |
| CN112630311B (en) | Metabolic markers and kits for detecting affective disorders and methods of use | |
| CN111562338B (en) | Application of transparent renal cell carcinoma metabolic marker in renal cell carcinoma early screening and diagnosis product | |
| US20160363560A9 (en) | Metabolite Biomarkers for the Detection of Esophageal Cancer Using NMR | |
| CN111279193B (en) | Behcet's disease diagnosis kit and method for detecting metabolite difference in urine | |
| CN112305121B (en) | Application of metabolic marker in atherosclerotic cerebral infarction | |
| JP2020517935A (en) | Diagnostic method for Behcet's disease using metabolite analysis | |
| CA3198282A1 (en) | Method of detecting lung cancer | |
| CN113552227A (en) | Combined markers for diagnosing childhood asthma and application and detection kit thereof | |
| CN113155983B (en) | Combined marker and application and detection kit thereof | |
| CN113552228A (en) | Combined markers for diagnosing childhood bronchiolitis and application and detection kit thereof | |
| CN114026427A (en) | Marker for diagnosing renal disease and diagnostic method | |
| CN112180007B (en) | Metabonomics-based diagnosis marker for generalized pustular psoriasis and application thereof | |
| CN112305119B (en) | Biomarker for atherosclerotic cerebral infarction and application thereof | |
| TWI721462B (en) | Non-alcoholic fatty liver disease detection method, non-alcoholic fatty liver disease detection kit and non-alcoholic fatty liver disease detection biomarker | |
| CN114047263A (en) | Application of metabolic marker in preparation of detection reagent or detection object for diagnosing AIS (automatic identification system) and kit | |
| CN113866285A (en) | Biomarker for diabetes diagnosis and application thereof | |
| CN111751457A (en) | Gouty arthritis diagnosis kit and application thereof | |
| CN114264767B (en) | Biomarkers for diabetes diagnosis and uses thereof | |
| CN117110493A (en) | Metabolic marker associated with neonatal pneumonia and metabolic acidosis and application thereof | |
| CN115825308A (en) | Application of nasopharyngeal carcinoma related urine marker in preparation of product for diagnosing/prognosing nasopharyngeal carcinoma |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20211026 |
|
| RJ01 | Rejection of invention patent application after publication |