CN109307764A - Application of one group of metabolic markers in terms of preparing diagnosis of glioma kit - Google Patents
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Abstract
The invention discloses application of one group of metabolic markers in terms of preparing diagnosis of glioma kit.It is a discovery of the invention that methylene-Pidolidone, allyl cysteine, capryl-l-carnitine and lauroyl-l-carnitine can combine for diagnosing glioma, diagnosis accuracy is high.Those skilled in the art can be developed collagen tumor early diagnosis kit; in the kit containing methylene-Pidolidone, allyl cysteine, capryl-l-carnitine and lauroyl-l-carnitine these four compounds standard items, for detecting methylene-Pidolidone in sample to be tested, allyl cysteine, capryl-l-carnitine and lauroyl-l-carnitine.The kit can also the standard items containing the chloro- L-phenylalanine of 2- be used as detection internal standard.
Description
Technical field
The invention belongs to biochemical fields, are related to application of the metabolic markers in medical diagnosis on disease, specific one group of metabolism
Application of the marker in terms of preparing diagnosis of glioma kit.
Background technique
Glioma (Glioma) is a kind of primary tumor originating from brain or spinal nerve spongiocyte, swollen in encephalic
It is most commonly seen in tumor, mostly in pernicious, account for the 80% of all malignant brain tumors.With the characteristics of glioma is grown by wettability, with surrounding group
It is fuzzy to knit boundary, has the characteristics that disease incidence height, high recurrence rate, the death rate are high and cure rate is low, seriously threatens human health.
Positive surgical operation chemoradiation therapy is still the main policies of current treatment glioma, and the rank of perform the operation prognosis and glioma
Or grade malignancy is closely related, therefore, early diagnosis and early intervention are the key that improve patient's prognosis and reduction case fatality rate.
Glioma early stage most incidence of occult, clinical symptoms are without specificity, and therefore, clinical diagnosis depends at present
The means such as the imageological examinations such as head CT, MRI and living tissue pathological examination.However, CT examination can not be distinguished well
Glioma and other brain lesions (such as inflammation, ischemic), and there is certain radiation hazradial bundle；And the inspection fee of MRI is more
It is expensive；The accuracy rate of diagnosis of biopsy is organized to be higher than imaging diagnosis, but the shadow of the factors such as heterogeneity, target area selection by tumour
It rings and still has certain misdiagnosis rate, and organizing biopsy is a kind of invasive diagnostic method.In short, a variety of factors limit these
Application of the diagnostic techniques in glioma early diagnosis and Mass screening.In recent years, isocitric dehydrogenase (IDH), tumour egg
The Molecular biomarkers such as white p53 (TP53), phosphate and tensin homologue (PTEN) are in glioma clinical diagnosis
Gradually paid attention to, but these markers are mostly to concentrate on gene and albumen level, sensitivity and specificity Shortcomings.
Therefore, it is very necessary to develop quick, noninvasive, effective glioma new diagnostic markers.
Summary of the invention
The present invention is directed to overcome the shortage of prior art, metabolic markers are provided in terms of preparing diagnosis of glioma kit
Using.
The object of the invention is achieved by following technical solution:
One group for diagnosing the metabolic markers of glioma, by methylene-Pidolidone, allyl cysteine, caprinoyl
Base-l-carnitine and lauroyl-l-carnitine composition.
Application of the above-mentioned metabolic markers in terms of preparing diagnosis of glioma kit.
Further, methylene-Pidolidone, allyl cysteine, capryl-l-carnitine are contained in the kit
With lauroyl-l-carnitine standard items.
Further, the standard items etc. in the kit also containing the chloro- L-phenylalanine of 2-.
Examine above-mentioned four kinds of metabolic markers to the accuracy rate of diagnosis of glioma by drawing ROC curve.As the result is shown:
In training set, methylene-Pidolidone, allyl cysteine, capryl-l-carnitine or lauroyl-l-carnitine are individually used for
When diagnosis of glioma, area (AUC) is respectively 0.797,0.809,0.804 and 0.776 under ROC curve, four marker joints
When for diagnosing, (sensitivity 88.0%, specificity is 93.5%) up to 0.959 by AUC；In test set, methylene-Pidolidone,
When allyl cysteine, capryl-l-carnitine or lauroyl-l-carnitine are individually used for diagnosis of glioma, area under ROC curve
It (AUC) is respectively 0.748,0.873,0.788 and 0.712, when four markers are combined for diagnosing, AUC is (sensitive up to 0.968
Degree 95.6%, specificity is 86.0%).One skilled in the art will appreciate that the area value in ROC curve evaluation method, under ROC curve
AUC, closer to 1, illustrates that diagnosis effect is better in the case where being greater than 0.5.AUC has lower accuracy at 0.5~0.7,
AUC has certain accuracy at 0.7~0.9, and AUC has high accuracy at 0.9 or more.It is verified, methylene-L- paddy ammonia
When acid, allyl cysteine, capryl-l-carnitine and lauroyl-l-carnitine are individually used for diagnosis of glioma, AUC is on 0.8 left side
The right side only has certain accuracy；When four use in conjunction, AUC is up to 0.96 or so, and accuracy is high.
In training set, methylene-Pidolidone, allyl cysteine, capryl-l-carnitine and lauroyl-L- meat
When four metabolic markers combinatorial association diagnosis of alkali, it is based on optimum sensitivity and specificity, obtains optimal critical value (Cut-
It offvalue) is 0.444.With the critical value carry out sample predictions, as the result is shown: this four kinds of metabolomes at metabolic indicator
Object group is 90.6% to the predictablity rate of training set, and the predictablity rate to test set is 90.5%.Accuracy rate is high.
The utility model has the advantages that
It is a discovery of the invention that methylene-Pidolidone, allyl cysteine, capryl-l-carnitine and lauroyl-L- meat
Alkali can combine for diagnosing glioma, and diagnosis accuracy is high.Those skilled in the art can be developed collagen tumor early stage
Diagnostic kit contains methylene-Pidolidone, allyl cysteine, capryl-l-carnitine and laurel in the kit
The standard items of these four compounds of acyl-l-carnitine, for detecting methylene-Pidolidone in sample to be tested, half Guang of allyl
Propylhomoserin, capryl-l-carnitine and lauroyl-l-carnitine.The kit can also be used containing the standard items of the chloro- L-phenylalanine of 2-
Make detection internal standard.
Detailed description of the invention
Fig. 1 is principal component analysis (PCA) shot chart of patients with gliomas vs healthy control group in embodiment；
Fig. 2 is the orthogonal partial least squares discriminant analysis (OPLS-DA) of patients with gliomas vs healthy control group in embodiment
Shot chart；
Fig. 3 is the permutation test figure for the OPLS-DA model that patients with gliomas vs healthy control group is established in embodiment；
Fig. 4 is embodiment methylene-Pidolidone, allyl cysteine, capryl-l-carnitine and lauroyl-L-
Four kinds of metabolic markers of carnitine individually and are used in combination the Receiver operating curve (ROC) point to training set diagnosis of glioma
Analysis；
Fig. 5 is embodiment methylene-Pidolidone, allyl cysteine, capryl-l-carnitine and lauroyl-L-
Four kinds of metabolic markers of carnitine individually and are used in combination the Receiver operating curve (ROC) point to test set diagnosis of glioma
Analysis；
Fig. 6 is embodiment methylene-Pidolidone, allyl cysteine, capryl-l-carnitine and lauroyl-L-
The accuracy rate of diagnosis proof diagram in training set to patients with gliomas is used in combination in four kinds of metabolic markers of carnitine；
Fig. 7 is embodiment methylene-Pidolidone, allyl cysteine, capryl-l-carnitine and lauroyl-L-
The accuracy rate of diagnosis proof diagram in test set to patients with gliomas is used in combination in four kinds of metabolic markers of carnitine.
Specific embodiment
It is specific with reference to the accompanying drawings and examples to introduce essentiality content of the present invention, but guarantor of the invention is not limited with this
Protect range.
One, laboratory apparatus and reagent
3000 ultra performance liquid chromatography system of Ultimate (Dionex company, the U.S.) series connection Q-Exactive level four bars-
Electrostatic field orbit trap high-resolution mass spectrometer (Thermo Fisher Scientific company, the U.S.)；Chromatographic column uses Waters
ACQUITY UPLC BEH C18 (50 × 2.1mm, 1.7 μm)；17 centrifuge of Heraeus Fresco (Thermo Fisher
Scientific)。
HPLC rank acetonitrile, methanol, formic acid are purchased from U.S. Thermo Fisher company；Experimental water is that Wahaha is pure
Water；The chloro- L-phenylalanine standard items of 2- are purchased from J&K Chemical (BeiJing, China).
Two, experimental method
1, experiment sample
After obtaining patient and agreeing to, foundation is diagnosed as with imageological examination result and postoperative pathological, collects in December, 2016
Patients with gliomas 95 in December, 2017 in the first affiliated hospital of Zhengzhou University income in hospital, all equal nothings of patients with gliomas
Other metabolic diseases.In addition, collecting healthy volunteer 96 from physical examination section is used as Normal group.All blood specimen collections are in trouble
Under person's early morning fasting state, each research object acquisition venous blood 3mL is placed in EDTA anticoagulant blood-collecting pipe, in 4 DEG C of items after acquisition
3000r/min is centrifuged 10min under part, draws supernatant (blood plasma), immediately in freezen protective in -80 DEG C of refrigerators after packing.From above
The training of 96 samples (including 46 Healthy People samples and 50 patients with gliomas samples) composition is randomly selected in 191 samples
Collection constructs diagnostic model for finding the difference metabolin of patients with gliomas and healthy population；95 samples of residue (including 50
Example Healthy People sample and 45 patients with gliomas samples) composition test set, for verifying difference metabolin as metabolic markers
Diagnose the ability of glioma.
2, sample preparation
Plasma sample is placed in after taking out to thaw on ice, is vortexed after mixing and draws 50 μ L plasma samples in 1.5mL centrifuge tube
In, the methanol solution (the chloro- L-phenylalanine 50ng/mL containing 2-) of 150 μ L containing the internal standards is added, is vortexed 30 seconds and mixes, 13000rpm
Be centrifuged 10min (4 DEG C), draw supernatant in sample introduction bottle to get.
Quality Control (QC) sample: it draws 5 μ L vortex respectively from all plasma samples and mixes, carried out according to above-mentioned same method
QC sample pre-treatments to get.In order to guarantee the reliability of data, QC sample analysis is interspersed in all sample metabolism group data and adopts
During collection, before sample analysis, 5 QC samples are continuously detected, start to analyze sample after instrument stabilizer, every 10 samples
Detect a QC solution.
3, in sample metabolin detection method
Chromatographic condition chromatographic isolation uses ultra performance liquid chromatography (UPLC, Waters Ultimate 3000, USA), color
Spectrum column be ACQUITY UPLC BEH C18 (50 × 2.1mm, 1.7 μm), 40 DEG C of column temperature.It is acetonitrile, B that flowing phase composition, which is A phase,
It is mutually 0.1% aqueous formic acid.Condition of gradient elution: 0~0.5min, 5%A；0.5~1.0min, 5%~60%A；0.5~
1.0min, 5%~60%A；1.0~7.0min, 60%~80%A；7.0~9.0min, 80%~100%A；9.0~
11.0min 100%A；11.0~13.0min, 5%A；Flow velocity 0.2mL/min, after column efflux do not shunt directly enter mass spectrum into
Row detection.
Mass Spectrometry Conditions mass spectral analysis uses Q Exactive level four bars-electrostatic field orbit trap high resolution mass spectrum, and ion source is
Heatable electric spray ion source (HESI).300 DEG C of temperature degree of auxiliary, it is 350 DEG C of ion source temperature, 320 DEG C of capillary temperature, auxiliary
Helping gas velocity is 10 μ L/min, and mass resolution 17,500, mass spectral analysis scan pattern: full scan/ddms2 scans model
Enclose m/z 80.00~1200.00.Impact energy gradient is 20,30 and 40eV.It is detected using positive ion mode, spray voltage
3.50kV, 40 μ L/min of sheath gas.All random sample introductions of sample, every 10 samples of detection are inserted into a needle blank, to avoid friendship
Fork pollution.
4, data processing and analysis
The data that Thermo Xcalibur 3.0 is collected pass through Sieve software (version 2.2, Thermo
Fisher Scientific) carry out peak extraction, peak alignment, peak correction, normalization etc. data pre-processings, output by sample names,
The three-dimensional data matrix of spectral peak information (including retention time and mass-to-charge ratio) and peak area composition.Data matrix is imported into SIMCA
(version14.0, Umetrics) carries out multi-variate statistical analysis.By establishing orthogonal partial least squares discriminant analysis (OPLS-
DA) model obtains variable weight importance ranking VIP (Variable Importance in the Projection) value.Choosing
Select to distinguish Difference of Metabolism between patients with gliomas and normal population contribute larger (VIP>1.5) and have significant changes (p<
0.05) difference metabolin of the metabolin as characterization glioma.
Through software CompoundDiscovererTM 2.1, structure dissection software Mass Frontier and mankind's metabolism group
The methods of the spectrum such as database (Human Metabolome Database, HMDB) library assisted retrieval is learned to difference endogenous metabolism
Object is identified, compares confirmation compound structure eventually by with standard items.
Using SPSS software (version 22.0, SPSS, Chicago, Illinois), pass through stepwise regression analysis, sieve
It selects with the factor that significantly affects as independent variable, establishes optimal regression equation, find out for distinguishing patients with gliomas and just
The potential source biomolecule marker of ordinary person group, returns and Receiver Operating Characteristics (Receiver Operator in conjunction with Logistic
Characteristic, ROC) diagnostic accuracy of the tracing analysis evaluation potential source biomolecule marker in training set and test set,
Susceptibility and specificity.The coordinate value taken when mounting index maximum value outstanding by ROC curve analytical calculation is Cut-off value, right
Test set sample is predicted, evaluates metabolic markers group to the predictive ability of glioma.
Three, experimental result
By principal component analysis (PCA) and building OPLS-DA model, (such as Fig. 1~3, the A in Fig. 1~3 represent training set, B
Represent verifying collection) as can be seen that in training set and test set, glioma group (Glioma, G) and Normal group (Normal,
H it) can be clearly separated, each self aggregation, show that the metabolism spectrum of patients with gliomas and healthy population has notable difference.In PCA figure
Preferably, the instrument and method for reflecting that this research uses are with good stability and repeated for the aggregation of QC sample.OPLS-DA's
Model evaluation parameter shows that model has good interpretability and predictive ability, using response rank test (RPT) method warp
It crosses 200 modelings and carries out displacement verifying, it was demonstrated that model stability is reliable, no over-fitting.
It is that standard screening goes out difference metabolin with VIP>1.5 and p<0.05, is further screened through SPSS stepwise regression analysis
4 metabolic markers are obtained, methylene-Pidolidone, allyl cysteine, capryl-l-carnitine and laurel are respectively as follows:
Acyl-l-carnitine, the results are shown in Table 1.Compared with normal group, expression water of above-mentioned 4 metabolic markers in patients with gliomas blood plasma
Average significant downward, changes multiple between 0.5-0.8 times, can be used as the potential diagnosis marker of glioma.
1 patients with gliomas blood plasma metabolic markers of table
Further examine above-mentioned four kinds of metabolic markers to the accuracy rate of diagnosis of glioma by drawing ROC curve.As a result
Display: in training set, methylene-Pidolidone, allyl cysteine, capryl-l-carnitine and lauroyl-l-carnitine list
A when being used for diagnosis of glioma, area (AUC) is respectively 0.797,0.809,0.804 and 0.776 under ROC curve, four marks
Internet of Things share when diagnosis, and (sensitivity 88.0%, specificity is 93.5%) up to 0.959 by AUC；In test set, methylene-L- paddy
When propylhomoserin, allyl cysteine, capryl-l-carnitine and lauroyl-l-carnitine are individually used for diagnosis of glioma, ROC curve
Lower area (AUC) is respectively 0.748,0.873,0.788 and 0.712, and when four markers are combined for diagnosing, AUC is up to 0.968
(sensitivity 95.6%, specificity is 86.0%).As a result as shown in Figures 4 and 5.
One skilled in the art will appreciate that the area value AUC under ROC curve is being greater than 0.5 in ROC curve evaluation method
In the case of, closer to 1, illustrate that diagnosis effect is better.AUC has lower accuracy at 0.5~0.7, and AUC is at 0.7~0.9
There is certain accuracy, AUC has high accuracy at 0.9 or more.It is verified, methylene-Pidolidone, half Guang ammonia of allyl
When acid, capryl-l-carnitine and lauroyl-l-carnitine are individually used for diagnosis of glioma, AUC only has one to fix 0.8 or so
True property；When four use in conjunction, AUC is up to 0.96 or so, and diagnosis accuracy is high.
In training set, methylene-Pidolidone, allyl cysteine, capryl-l-carnitine and lauroyl-L- meat
When four metabolic markers combinatorial association diagnosis of alkali, it is based on optimum sensitivity and specificity, obtains optimal critical value (Cut-
It offvalue) is 0.444.With the critical value carry out sample predictions, as the result is shown: this four kinds of metabolomes at metabolic indicator
Object group is 90.6% to the predictablity rate of training set, and the predictablity rate to test set is 90.5%.As a result such as the institute of Fig. 6~7
Show.
To sum up, methylene-Pidolidone, allyl cysteine, capryl-l-carnitine and lauroyl-l-carnitine
It can combine for diagnosing glioma, diagnosis accuracy is high.Those skilled in the art can be developed collagen tumor and be examined in early days
Break kit, contains methylene-Pidolidone, allyl cysteine, capryl-l-carnitine and lauroyl-in the kit
The standard items of these four compounds of l-carnitine, for detecting methylene-Pidolidone in sample to be tested, half Guang ammonia of allyl
Acid, capryl-l-carnitine and lauroyl-l-carnitine.The kit can also be used as containing the standard items of the chloro- L-phenylalanine of 2-
Detect internal standard.
The effect of above-described embodiment is specifically to introduce essentiality content of the invention, but those skilled in the art should know
Protection scope of the present invention should not be confined to the specific embodiment by road.
Claims (4)
1. one group for diagnosing the metabolic markers of glioma, it is characterised in that: by methylene-Pidolidone, half Guang of allyl
Propylhomoserin, capryl-l-carnitine and lauroyl-l-carnitine composition.
2. application of the metabolic markers described in claim 1 in terms of preparing diagnosis of glioma kit.
3. application according to claim 2, it is characterised in that: contain methylene-Pidolidone, allyl in the kit
Base cysteine, capryl-l-carnitine and lauroyl-l-carnitine standard items.
4. application according to claim 3, it is characterised in that: also containing the chloro- L-phenylalanine of 2- in the kit
Standard items.
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| CN111929399A (en) * | 2019-10-12 | 2020-11-13 | 北京航空航天大学 | Urine metabolic marker of glioblastoma patient carrying IDH gene mutation and application thereof |
| CN112201356A (en) * | 2020-10-09 | 2021-01-08 | 郑州大学第一附属医院 | Construction method of oral squamous cell carcinoma diagnosis model, marker and application thereof |
| CN113984935A (en) * | 2021-11-17 | 2022-01-28 | 东莞理工学院 | Method for researching metabolic characteristics of acetoacidophilic proteophilus based on metabolome analysis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111929399A (en) * | 2019-10-12 | 2020-11-13 | 北京航空航天大学 | Urine metabolic marker of glioblastoma patient carrying IDH gene mutation and application thereof |
| CN111929399B (en) * | 2019-10-12 | 2022-09-30 | 北京航空航天大学 | Urine metabolic marker of glioblastoma patient carrying IDH gene mutation and application thereof |
| CN112201356A (en) * | 2020-10-09 | 2021-01-08 | 郑州大学第一附属医院 | Construction method of oral squamous cell carcinoma diagnosis model, marker and application thereof |
| CN112201356B (en) * | 2020-10-09 | 2022-02-01 | 郑州大学第一附属医院 | Construction method of oral squamous cell carcinoma diagnosis model, marker and application thereof |
| CN113984935A (en) * | 2021-11-17 | 2022-01-28 | 东莞理工学院 | Method for researching metabolic characteristics of acetoacidophilic proteophilus based on metabolome analysis |
| CN113984935B (en) * | 2021-11-17 | 2024-04-02 | 东莞理工学院 | Method for researching metabolic characteristics of acetoacidophile based on metabonomic analysis |
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