CN114280202B - Biomarker for diagnosing cadmium poisoning and application thereof - Google Patents
Biomarker for diagnosing cadmium poisoning and application thereof Download PDFInfo
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Abstract
The application relates to the technical field of heavy metal poisoning detection, in particular to a biomarker for diagnosing cadmium poisoning and application thereof. The biomarker is N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide, and a cadmium poisoning detection system is also constructed by using the biomarker. The technical problem that the diagnosis index reflecting the organism metabolic condition of the cadmium poisoning patient is lacking in the prior art can be solved. Through analysis of working characteristic curves of subjects, detection of a large number of clinical samples and correlation analysis of urine cadmium levels, the N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide of the scheme can reflect cadmium exposure levels of sample groups more sensitively, and can be used as a biomarker for reflecting metabolic conditions of the groups to be applied to detection and diagnosis of cadmium poisoning.
Description
Technical Field
The application relates to the technical field of heavy metal poisoning detection, in particular to a biomarker for diagnosing cadmium poisoning and application thereof.
Background
Cadmium is a soft metal whose dust, fumes and vapors are mainly inhaled into the body through the respiratory tract. Cadmium in the body is mainly accumulated in the kidney and liver in the form of metallothionein, and is slowly excreted. Occupational acute poisoning is commonly seen by inhalation of high concentrations of cadmium oxide soot. Chronic poisoning can occur by workers working for a long time with cadmium smelting and applying cadmium and its compounds, if the workers do not pay attention to protection. Acute poisoning of raw active cadmium poisoning is usually taken by mistaking cadmium salt or placing an acidic diet in a cadmium plating vessel. The chronic poisoning of raw active cadmium is caused by long-term living in cadmium-polluted areas, or long-term use or taking of water, rice or tobacco polluted by cadmium. Cadmium poisoning can cause kidney toxicity, bone injury, neurotoxicity, cardiovascular injury, diabetes, cancer and the like, and seriously affects the health of people.
In 1987, national promulgates a diagnostic standard and a treatment principle for occupational cadmium poisoning (GB 7803-1987). The 2002 edition is updated to GBZ 17-2002, and the revised standard divides acute cadmium poisoning into light, medium and heavy three stages so as to guide clinical emergency work; the diagnostic value of the urine cadmium and urine beta 2-microglobulin of the chronic cadmium poisoning is changed into a unit for creatinine correction, so that the currently unusual urine protein electrophoresis examination index is eliminated, the urine retinol binding protein measurement index is increased, and the diagnosis of the chronic mild cadmium poisoning is more reasonable and easy to master. The currently adopted national standard version GBZ-2015 of occupational cadmium poisoning is completely consistent with the earlier 2002 version.
However, cadmium poisoning is defined jointly from a physical and biochemical perspective with urine cadmium concentration and beta 2-microglobulin concentration as diagnostic. In practical operation, the two indexes need to be evaluated by combining clinical symptoms, but the clinical symptoms of cadmium poisoning of patients are greatly different, and the clinical symptoms of some patients are not obvious. The combination of the urine cadmium concentration index and the beta 2-microglobulin concentration index with clinical needle-shaped diagnosis of cadmium poisoning has certain limitation. There is a need to find a new index or marker that can more accurately reflect the cadmium poisoning level of a patient, so as to overcome the problems existing in the existing clinical diagnosis of cadmium poisoning, so as to more accurately reflect the metabolic condition of the organism during cadmium poisoning, thereby increasing the diagnosis accuracy.
Disclosure of Invention
The application aims to provide a biomarker for diagnosing cadmium poisoning, which aims to solve the technical problem that the prior art lacks diagnostic indexes reflecting the body metabolic condition of a patient suffering from cadmium poisoning.
In order to achieve the above purpose, the application adopts the following technical scheme:
a biomarker for diagnosing cadmium poisoning, which is N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide.
The scheme also provides application of the biomarker for diagnosing cadmium poisoning in construction of a cadmium poisoning detection system, wherein the cadmium poisoning detection system is used for detecting N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide.
The principle and the advantages of the scheme are as follows:
the technical scheme combines with metabonomics analysis technology, and searches the metabolic marker N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide closely related to cadmium poisoning by examining the changes of metabolites in urine of patients with cadmium poisoning and non-poisoning patients. The content level of the compound in the cadmium poisoning group and the non-poisoning group is compared, the content is increased by 9.594 times, and the compound has potential clinical application value.
A biomarker is an endogenous substance that can be objectively detected and evaluated, and can be used as an indicator of normal biological processes, pathological processes or therapeutic intervention pharmacological responses, and finding valuable biomarkers has become a research hotspot in the current medical field. Currently, countries have clear diagnostic indicators (urinary cadmium concentration and beta 2-microglobulin concentration) in terms of cadmium poisoning. However, the use of urinary cadmium concentration and beta 2-microglobulin concentration as a diagnosis is mainly defined by the combination of physical and biochemical aspects of cadmium poisoning, and needs to be evaluated in combination with clinical symptoms. However, clinical symptoms are often unstable, and clinical manifestations of different patients are greatly different. The technical scheme screens and identifies the marked metabolic products of the organism to evaluate the cadmium poisoning state, the metabolic products can reflect the physiological state of the organism more sensitively, are closely related to the condition of the organism, and can reflect the cadmium poisoning condition of patients more objectively compared with clinical symptoms. The inventors performed efficacy assessment on the novel metabolic marker N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide. The diagnostic and prognostic effect of the use of the present markers on the whole is evaluated by analysis of the subject's working characteristics and calculation of the youden about log index. As a result, N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide is found to show very high diagnostic ability, and sensitivity, specificity and accuracy are all very close to 100%.
In addition, the inventor confirms that the content of N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide in the cadmium poisoning group is obviously higher than that in urine of the control group population through detecting a large number of clinical samples. And with the increase of cadmium concentration, the level of N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide in urine is also increased, the correlation R value reaches 0.722 (p < 0.0001), and the positive correlation is obvious. The inventor also analyzes the working characteristic curve of the subjects aiming at large sample population, the AUC value of the working characteristic curve is up to 0.8916, the AUC value shows that the diagnostic effect of the N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide serving as a marker is very excellent, and the high clinical application value is shown. And the AUC values of the large sample study were very close compared to the AUC values (0.911) previously obtained in the 15 pairs of samples, further confirming that the marker can stably reflect cadmium exposure of the sample population.
The above research results prove that the N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide can more sensitively reflect the cadmium exposure level of sample populations, and can be used as a biomarker for reflecting the metabolic conditions of the sample populations to be applied to the detection and diagnosis of cadmium poisoning.
Further, the diagnostic reference value in urine was 0.06176. Mu.g/L. The diagnosis reference value of N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide in urine is 0.06176 mug/L determined by large sample experiment and ROC analysis, and the result is exceeding the value, and the result can be preliminarily determined to be cadmium poisoning.
Further, the cadmium poisoning detection system comprises a detection unit and an extraction reagent for extracting metabolites in a sample; the detection unit comprises a liquid chromatography-mass spectrometry device.
The technical scheme uses liquid chromatography-mass spectrometry equipment to analyze N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide in a sample. The liquid chromatography-mass spectrometry combined device is an instrument combining liquid chromatography and mass spectrometry and is mainly used for analyzing substances which cannot be analyzed by GC/MS or have poor thermal stability, strong polarity and high molecular weight, such as biological samples (medicines and metabolites thereof) and biological macromolecules (peptides, proteins, nucleic acids and polysaccharides).
Further, the sample is urine.
The technical scheme can accurately detect the N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide in the urine sample and eliminate the interference of other components in the urine sample. In addition, urine detection has the advantages of simple and convenient sample collection, no wound, huge physiological and pathological information quantity and the like, and is beneficial to disease diagnosis, treatment, tracking and monitoring of prognosis processes and the like.
Further, the reagent for pretreating a sample includes a methanol-acetonitrile mixed solvent and a methanol-water mixed solvent.
Further, the metabolites in the sample are extracted by the following method: adding an internal standard and a methanol-acetonitrile mixed solvent into a sample, performing vortex oscillation and ice water bath ultrasonic extraction, standing, and centrifuging to obtain a supernatant to obtain an extract A; after the organic components in the extracting solution A volatilize, adding a methanol-water mixed solvent, carrying out vortex vibration and ice water bath ultrasonic extraction, standing, and centrifuging to obtain a supernatant to obtain an extracting solution B; and filtering the extracting solution B to obtain a to-be-detected product.
By the extraction method, impurities such as protein, cells and the like in the urine sample can be removed, and the to-be-detected substances in the sample can be enriched, so that macromolecular substances can be prevented from blocking equipment, and the detection sensitivity of N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide can be improved.
Further, the methanol-acetonitrile mixed solvent consists of methanol and acetonitrile in a volume ratio of 2:1; the methanol-water mixed solvent consists of methanol and water in a volume ratio of 1:4.
By adopting the reagent with the volume ratio, metabolites in urine can be fully extracted from the urine and interfering substances can be removed, so that the detection accuracy is improved, the matrix effect is reduced, and the detection sensitivity is improved.
Further, the liquid chromatography conditions of the liquid chromatography-mass spectrometry combined equipment are as follows:
chromatographic column: ACQUITY UPLC HSS T3, 100mm×2.1mm,1.8 μm;
column temperature: 45 ℃;
mobile phase: water containing 0.1% formic acid as mobile phase a, acetonitrile containing 0.1% formic acid as mobile phase B;
elution mode: gradient elution;
flow rate: 0.35mL/min;
sample injection volume: 2. Mu.L.
By adopting the chromatographic conditions, the target substance N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide can be separated from other interfering substances, and the target substance is further enriched for subsequent mass spectrum detection.
Further, the ion source of the liquid chromatography-mass spectrometry combined equipment is an electron bombardment source, and a positive and negative ion scanning mode is adopted for mass spectrum signal acquisition; the ion source parameters are as follows: the electrospray voltage of positive ions is 3800V, and the electrospray voltage of negative ions is-3000V; the capillary temperature was 320 ℃; the temperature of the auxiliary gas is 350 ℃; the sheath gas flow rate is 35Arb; the flow rate of the auxiliary gas is 8Arb; the mass spectrometry scan parameters were as follows: the mass scanning range is 100-1200m/z; the full scan resolution is 70000; the resolution of the secondary mass spectrum was 17500.
The electron bombardment ion source uses high-speed (high-energy) electron beam to impact the sample, so as to generate electrons and molecular ions, and the molecular ions are continuously subjected to electron bombardment to cause the rupture of chemical bonds or the molecular rearrangement, so that various ions are instantaneously generated. The mass spectrogram obtained by using the electron bombardment source has good reproducibility and contains more fragment ion information, and is helpful for presumption of the structure. By adopting the mass spectrum conditions, ideal atomization and ionization effects can be obtained, the optimal atomization conditions are achieved, and the sensitivity is improved.
Drawings
FIG. 1 shows the result of PLS-DA analysis of example 1.
FIG. 2 is a volcanic plot of the results of metabonomics analysis of example 1.
FIG. 3 is a graph showing the result of ROC analysis in example 2.
FIG. 4 is a typical chromatogram and mass spectrum of the N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide standard of example 3.
FIG. 5 is a typical chromatogram of a urine sample of example 3 and a mass spectrum of N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide.
FIG. 6 is the statistics of the results of the differential expression analysis of the control group and the cadmium poisoning group of example 3.
FIG. 7 is a graph showing the results of correlation analysis between the marker content and the cadmium urine content in example 3.
Fig. 8 is a graph of the ROC analysis results for the large sample of example 3.
Detailed Description
The present application will be described in further detail with reference to examples, but embodiments of the present application are not limited thereto. Unless otherwise indicated, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used are all commercially available.
Example 1: urine metabonomics analysis and potential biomarker screening for cadmium poisoning patients
(1) Sample case
A representative population 30 who met the national cadmium poisoning standard and failed to meet the poisoning standard was collected (diagnostic standard reference national standard GBZ-2015). Age range: women aged 40-50, control group 15, patient group 15, see Table 1 for details.
Table 1: detailed information of cadmium poisoning patient group and control group (average value in brackets)
| Cadmium poisoning group | Control group | |
| Quantity of | 15 | 15 |
| Sex (sex) | Female woman | Female woman |
| Age (year) | 42-50(47) | 40-49(45) |
| Work age (years) | 8-20(10) | 5-18(7) |
| Urine cadmium concentration (μg/g creatinine) | 12.0-26.0(17.06) | 0.7-4.9(3.8) |
| Beta-2 microglobulin (mug/g creatinine) | 1129.4-3718.2(1785.71) | 20.3-251.5(161.013) |
(2) Metabonomics analysis based on LC-MS (liquid chromatography-mass spectrometry)
The LC-MS procedure includes: sample pretreatment, metabolite extraction, liquid chromatography-mass spectrometry analysis, data processing, statistical analysis and the like, and the specific processes are as follows:
(2.1) sample pretreatment and metabolite extraction
Thawing urine samples of the sample population on ice, taking 100 mu L of urine samples, and adding 10 mu L of internal standard (L-2-phenylalanine, 0.06mg/mL; methanol configuration); adding 300 mu L of a methanol-acetonitrile mixed solvent (V: V=2:1), and vortex shaking for 1min; ultrasonic extracting with ice water bath for 10min, standing at-20deg.C for 30min; centrifuging for 10min (13000 rpm,4 ℃) collecting 350 μl supernatant, placing into LC-MS sample injection vial, and volatilizing; redissolved with 300 μl of methanol-water mixed solvent (V: v=1:4) (vortexed for 30s, sonicated for 3 min); standing at-20 ℃ for 2 hours; after centrifugation for 10min (13000 rpm,4 ℃) 150. Mu.L of the supernatant was aspirated by a syringe and filtered through a 0.22 μm organic phase pinhole filter, the extract (sample to be tested) of the sample was obtained. The extract was transferred to an LC-MS sample vial and stored at-80 ℃ until LC-MS analysis was performed. The quality control sample (QC) is prepared by mixing all the extracting solutions of the samples in equal volume. In the whole extraction process, all the extraction reagents are pre-cooled at-20 ℃ before use.
(2.2) liquid chromatography-Mass Spectrometry analysis
The analytical instrument used in this example was a liquid-mass combination system consisting of a Dionex U3000 UHPLC ultra high performance liquid chromatography tandem QE plus high resolution mass spectrometer.
The chromatographic conditions were as follows:
chromatographic column: ACQUITY UPLC HSS T3 (100 mm. Times.2.1 mm,1.8 μm);
column temperature: 45 ℃;
mobile phase: mobile phase a-water (0.1% formic acid by volume) mobile phase B-acetonitrile (0.1% formic acid by volume);
elution mode: gradient elution (elution procedure see table 2);
flow rate: 0.35mL/min;
sample injection volume: 2. Mu.L.
The elution procedure is shown in table 2.
Table 2: gradient elution procedure
| Time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 95 | 5 |
| 2 | 95 | 5 |
| 4 | 70 | 30 |
| 8 | 50 | 50 |
| 10 | 20 | 80 |
| 14 | 0 | 100 |
| 15 | 0 | 100 |
| 15.1 | 95 | 5 |
| 16 | 95 | 5 |
The mass spectrometry conditions were as follows:
ion source: ESI; the sample mass spectrum signal acquisition adopts a positive and negative ion scanning mode respectively, and mass spectrum parameters are shown in Table 3 in detail.
Table 3: mass spectral parameters
(3) Data processing and statistical analysis: searching urine differential metabolites of cadmium poisoning patients and non-poisoning patients by using multivariate statistics
The Orthogonal Signal Correction (OSC) and PLS-DA methods are combined using orthogonal partial least squares discriminant analysis (OPLS-DA), and the variance variable is screened by removing uncorrelated variance. VIP values are projected variable importance of the first major component of PLS-DA, and thus analyzed for changes in metabolite expression in urine of patients with cadmium poisoning and non-poisoning, as shown in FIG. 1. VIP > 1 is generally used as a common evaluation standard of metabonomics and is used as one of the standards for differential metabolite screening. Fig. 1 is a graph of the score obtained by dimension reduction of the first principal component and the second principal component in the cadmium poisoning group and the non-poisoning group, the abscissa represents the inter-group difference, the ordinate represents the intra-group difference, and the two groups of results are well separated, indicating that this scheme can be used. Fig. 2 is a volcanic chart, the abscissa represents log2 log of the fold difference, and the ordinate represents the result of T test (negative log10 log of p value), which is used to evaluate whether the difference between two groups of samples is significant, and preliminary screening is performed. Meanwhile, under the conditions that p is more than 0.05 and VIP is more than 1, 662 differences exist between the cadmium poisoning group and the control group. Ranking with VIP values, the first candidate biomarker ranked for cadmium-poisoned patients was further selected. The first marker of the sequence was N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide (see formula (1), CAS number 1695-02-9). See table 4 for specific information on this marker. As can be seen from comparative analysis of the sample information, the content of N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide in the cadmium poisoning group is increased by 9.594 times compared with that in the non-poisoning group.
Table 4: specific information for LC-MS identified biomarkers
Example 2: urine biomarker analysis and subject work characteristic curve analysis for cadmium poisoning patient
The working characteristic curve (receiver operating characteristic curve, abbreviated as ROC curve) of the subject, also called as susceptibility curve (sensitivity curve), is used for judging the quality of classification and detection results. ROC curves are very important and common statistical analysis methods. Is a graph formed by taking a false positive rate (False positive rate, 1-specificity) as a horizontal axis and a true positive rate (True positive rate, sensitivity) as a vertical axis, and is a curve drawn by different results obtained by testing samples according to different judgment standards (thresholds). The youden about log index calculation was performed on the metabonomics data of this protocol to reflect the overall diagnostic and predictive effect of a single index (N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide). Using metabonomic analysis data, the relative content of the biomarker was used as a diagnostic variable, the sensitivity and specificity at specific values for each diagnostic variable was calculated, and ROC curves were plotted, with results shown in fig. 3. The AUC values, specificity and sensitivity results at the youden about dendriet optimum cut-off are shown in table 4. The area under the curve AUC is used to represent accuracy, the higher the AUC value, the higher the accuracy, whereas the AUC value for diagnosing cadmium poisoning using the marker of the present scheme reaches 0.911. Sensitivity reflects the true positive rate of cadmium poisoning diagnosis by using the marker of the scheme, and specificity reflects the true negative rate of cadmium poisoning diagnosis by using the marker of the scheme. The data show that N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide has very high diagnostic capability, sensitivity, specificity and accuracy are all very close to 100%, and can be applied to clinical detection in future.
Example 3: clinical sample detection, biomarker effectiveness verification, and subject work characteristic curve analysis
In the embodiment, 50 urine samples of a cadmium poisoning sample group and 50 urine samples of a control group sample group are collected, and the content of N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide in the sample is detected and the correlation between the N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide and cadmium poisoning is analyzed by utilizing a liquid chromatography-mass spectrometry technology. And subject work characteristic curve analysis (ROC curve) analysis was performed on the test results of the 100 cases of urine.
(1) LC-MS detection
Thawing urine samples of the sample population on ice, taking 100 mu L of urine samples, and adding 10 mu L of internal standard (L-2-phenylalanine, 0.06mg/mL; methanol configuration); adding 300 mu L of a methanol-acetonitrile mixed solvent (V: V=2:1), and vortex shaking for 1min; ultrasonic extracting with ice water bath for 10min, standing at-20deg.C for 30min; centrifuging for 10min (13000 rpm,4 ℃) collecting 350 μl supernatant, placing into LC-MS sample injection vial, and volatilizing; redissolved with 300 μl of methanol-water mixed solvent (V: v=1:4) (vortexed for 30s, sonicated for 3 min); standing at-20 ℃ for 2 hours; after centrifugation for 10min (13000 rpm,4 ℃) 150. Mu.L of the supernatant was aspirated by a syringe and filtered using a 0.22 μm organic phase pinhole filter, an extract of the sample was obtained. The extract was transferred to an LC-MS sample vial and stored at-80 ℃ until LC-MS analysis was performed. In the whole extraction process, all the extraction reagents are pre-cooled at-20 ℃ before use.
The chromatographic and mass spectrometric conditions of this example were the same as in example 1 and characterized by the amount of the marker per L of urine (μg) using N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide standard (SIGMA-RBI Co., ltd.; cat# 9970) to draw a standard curve and calculating the N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide content of the urine sample by the standard curve, using the methods conventional in the art. Under the same chromatographic and mass spectrum conditions, carrying out LC-MS detection on a standard solution prepared from the N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide standard and a urine sample, wherein a chromatographic peak with the same retention time as that of the N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide peak in the standard solution appears in the chromatogram of the urine sample, namely the chromatographic peak of the N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide in the urine sample is judged. Typical chromatograms of the N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide standard (retention time 2.55 min) and mass chromatograms of the N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide are shown in FIG. 4, and typical chromatograms of the urine sample (retention time 2.54min of the N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide) and mass chromatograms of the N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide in the sample are shown in FIG. 5.
(2) Comparison of differences of N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide in different samples
As can be seen from the comparison result of the content of N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide in urine of the control group and the cadmium poisoning group, the content of N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide in the cadmium poisoning group is obviously higher than that of urine of the control group, and the marker can reflect the cadmium exposure condition of the sample group, so that the detection and diagnosis of cadmium poisoning are realized.
(3) Correlation analysis of N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide content and urine cadmium content
In addition, for the content level of the marker in urine, we also analyze the correlation of the marker and the cadmium level in urine, and referring to fig. 7, the analysis result of the correlation shows that as the cadmium concentration is increased, the level of N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide in urine is also increased, the correlation R value reaches 0.722 (p < 0.0001), and the positive correlation is obvious. The N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide can reflect the cadmium exposure level of a sample population more sensitively, and can be used as a biomarker for reflecting the metabolic condition of the sample population to be applied to the detection and diagnosis of cadmium poisoning.
(4) Subject work characteristic analysis for large samples
To further verify the application value of N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide as a biomarker of cadmium poisoning, we plotted ROC curve with the content of N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide in urine as diagnostic variable for the test results of 100 samples of this experimental example, see FIG. 8. The AUC value is 0.8916, which is taken as a marker and is a very excellent parameter, and the AUC value shows higher clinical application value. And very close to the AUC value (0.911) previously obtained in the 15 pairs of samples, it was further demonstrated that the marker reflects and is more stable in the cadmium exposure of the sample population. According to the ROC curve, the value of the point with the best sensitivity and specificity is selected as the critical value of diagnosing cadmium poisoning by N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide, which is 0.06176 mug/L, and the parameter can be further referred to realize the detection and auxiliary diagnosis of cadmium poisoning.
The foregoing is merely exemplary of the present application, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present application, and these should also be regarded as the protection scope of the present application, which does not affect the effect of the implementation of the present application and the practical applicability of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (7)
1. An application of a urine biomarker for diagnosing cadmium poisoning in constructing a cadmium poisoning detection system, which is characterized in that: the urine biomarker is N- (2, 4-dinitrophenyl) -DL-methionine sulfoxide; the cadmium poisoning detection system comprises a detection unit and an extraction reagent for extracting metabolites in a sample; the detection unit comprises a liquid chromatography-mass spectrometry device.
2. The use of a urine biomarker for diagnosing cadmium poisoning according to claim 1 in constructing a cadmium poisoning detection system, wherein: its diagnostic reference value in urine was 0.06176. Mu.g/L.
3. Use of a urine biomarker for diagnosing cadmium poisoning according to claim 2 in the construction of a cadmium poisoning detection system, characterized in that: the extraction reagent comprises a methanol-acetonitrile mixed solvent and a methanol-water mixed solvent.
4. Use of a urine biomarker for diagnosing cadmium poisoning according to claim 3 in the construction of a cadmium poisoning detection system, characterized in that: the metabolites in the sample were extracted by the following method: adding an internal standard and a methanol-acetonitrile mixed solvent into a sample, performing vortex oscillation and ice water bath ultrasonic extraction, standing, and centrifuging to obtain a supernatant to obtain an extract A; after the organic components in the extracting solution A volatilize, adding a methanol-water mixed solvent, carrying out vortex vibration and ice water bath ultrasonic extraction, standing, and centrifuging to obtain a supernatant to obtain an extracting solution B; and filtering the extracting solution B to obtain a to-be-detected product.
5. The use of a urine biomarker for diagnosing cadmium poisoning according to claim 4 in constructing a cadmium poisoning detection system, wherein: the methanol-acetonitrile mixed solvent consists of methanol and acetonitrile in a volume ratio of 2:1; the methanol-water mixed solvent consists of methanol and water in a volume ratio of 1:4.
6. The use of a urine biomarker for diagnosing cadmium poisoning according to claim 5 in constructing a cadmium poisoning detection system, wherein: the liquid chromatography conditions of the liquid chromatography-mass spectrometry equipment are as follows:
chromatographic column: ACQUITY UPLC HSS T3, 100mm×2.1mm,1.8 μm;
column temperature: 45 ℃;
mobile phase: water containing 0.1% formic acid as mobile phase a, acetonitrile containing 0.1% formic acid as mobile phase B;
elution mode: gradient elution;
flow rate: 0.35mL/min;
sample injection volume: 2. Mu.L.
7. The use of a urine biomarker for diagnosing cadmium poisoning according to claim 6 in constructing a cadmium poisoning detection system, wherein: the ion source of the liquid chromatography-mass spectrometry equipment is an electron bombardment source, and a positive and negative ion scanning mode is adopted for mass spectrum signal acquisition; the ion source parameters are as follows: the electrospray voltage of positive ions is 3800V, and the electrospray voltage of negative ions is-3000V; the capillary temperature was 320 ℃; the temperature of the auxiliary gas is 350 ℃; the sheath gas flow rate is 35Arb; the flow rate of the auxiliary gas is 8Arb; the mass spectrometry scan parameters were as follows: the mass scanning range is 100-1200m/z; the full scan resolution is 70000; the resolution of the secondary mass spectrum was 17500.
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| CN104267091A (en) * | 2014-09-15 | 2015-01-07 | 江苏大学 | Method for detecting cadmium poisoning of organisms by virtue of serum biomarker protein |
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