CN106370744A - Application of 8-hydroxydeoxyguanosine as urine marker - Google Patents
Application of 8-hydroxydeoxyguanosine as urine marker Download PDFInfo
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Abstract
The invention provides an application of 8-hydroxydeoxyguanosine as a urine marker. The molecular formula of the 8-hydroxydeoxyguanosine is C10H13O5N5. In detection, collection of a sample is non-invasive, and the sample is easy to obtain and is large in acquisition quantity. Compared with FOBT test, the method is more acceptable for detectors and can reduce mental burden of work staff, and is more acceptable for common people. By means of optimized off-line solid-phase extraction technology with ultrahigh performance liquid chromatography-tandem mass spectrometry, the concentration of a DNA oxidative damage marker, 8-hydroxydeoxyguanosine, in urine of a to-be-tested subject can be accurately quantified, so that disease risk analysis to the subject can be carried out through an established colorectal cancer incidence prediction model. The urine marker, when being used for screening the colorectal cancer, is non-invasive, quick and high-flux. The detection method not only is suitable for early stage detection of the colorectal cancer but also is suitable for detection of metastasis or non-metastasis of the colorectal cancer. The application has excellent prospect.
Description
Technical field
The invention belongs to detection technique field, it is related to malignant tumor detection technique, more particularly, to a kind of colorectal cancer early stage
Examination urine markers thing and its application, are a kind of new Noninvasive screening methods, and in vitro colorectal cancer is carried out with early stage
Find and detection.
Background technology
Colorectal cancer is a kind of common malignant tumor, is that sickness rate rises one of tumor the fastest in recent years.In the world
In the range of sickness rate male be the 3rd, women be second.With expanding economy, growth in the living standard and life side
The change of formula, its sickness rate is also by the continuous trend rising.Although the diagnosis of colorectal cancer in recent years and treatment have very big
Progress, but the general curative effect of colorectal cancer being not improved, this mainly due in the patient going to see a doctor at present, one
More than half has been late period.And early stage colorectal cancer patients prognosis is preferably, 5 years survival rates of Post operation may be up to more than 90%, and late period
Then less than 10%.Early discovery, early diagnosis and early treatment are one of important control strategies of colorectal cancer.But when being at early stage,
The usual non-evident sympton of patient, also will not carry out hospital admission.At present, the early diagnostic rate of colorectal cancer is very low, improves it early
Phase diagnosis will be obviously improved the overall treatment effect of colorectal cancer.
At present, screening is mainly used in the examination of China's colorectal cancer, is joined using high risk factor questionnaire survey
Close fecal occult blood detection (fobt).The examination crowd that questionnaire survey is positive or fobt is positive, accepts enteroscopy.But feces are dived
Blood loss is very high, and not all bleeder is colorectal cancer, and false-positive appearance can increase and be accepted knot by inspection individuality
The misery of the inspections such as intestinal mirror.Additionally, feces can cause vision and psychological burden to tester.
For feces, urine is relatively easily detected personnel and accepts, additionally, other bio-matrix phases with serum etc.
Urine has the characteristics that noinvasive sampling, is easily obtained substantially product sample ratio, is especially suitable for clinical research and the reality of great amount of samples
Trample.Thus, the colorectal cancer methods for screening based on urine specimen for the exploitation, the inspection that can not only mitigate the masses is painful, also will
The early stage contributing to colorectal cancer precisely finds and diagnoses.
Substantial amounts of inorganic salt, such as sodium ion, potassium ion, chloride ion is contained, solid phase extraction techniques can be effectively right in urine
Urine specimen carries out desalination, concentrates and is enriched with.Ultra Performance Liquid Chromatography mass spectrometric hyphenated technique combines the powerful separation energy of chromatograph
Power and the excellent quantitation of mass spectrum and Structural Identification function, are widely used in numerous neck such as chemistry, life sciences, health care
Domain.
8-OhdG (8-ohdg), guanine in active oxygen species such as hydroxide radicals attack dna chain
The 8th product that carbon atom is generated of base, is usually used to the degree of assessment organism dna oxidative damage.The dna damaging exists
After a series of repair mechanism, 8-OhdG is excised on dna chain, and is finally excreted in urine.
Content of the invention
It is an object of the invention to provide a kind of 8-OhdG (8-ohdg) is as the application of urine detection mark,
The molecular formula of described 8-OhdG (8-ohdg): c10h13o5n5, structural formula:
8-OhdG of the present invention, as the application of mark, is realized by following steps:
1. the pretreatment of sample: urine specimen to be measured is stored in -80 DEG C of refrigerators, takes out solution at 4 DEG C before experiment
Freeze, take after centrifugation 0.5ml supernatant add 0.5ml water dilution, and add 10pmol Isotopic Internal Standard [15n5] 8-ohdg, described
Solid-Phase Extraction is to add 1.0ml methanol to be activated in hlb oasis pillar, adds 1.0ml water and is balanced, adds
Urine specimen, is subsequently carried out with 1.0ml 5% methanol, finally carries out eluting with 1.0ml 50% methanol, collects eluent
And be dried with vacuum centrifuge, subsequently add the redissolution of 0.2ml water to be measured；
2. the urine after Solid-Phase Extraction pre-treatment carries out separating using liquid chromatograph: for making complicated group in urine specimen
Divide especially guanosine can separate with 8-OhdG as much as possible, chromatographic condition used is: chromatographic column is waters
Beh c18 post (1.7 μm, 2.1 × 100mm), column temperature be 40 DEG C, mobile phase be 0.1% acetic acid aqueous solution-methanol (92.5:7.5,
V/v), flow velocity is 0.25ml/min, and applied sample amount is 5 microlitres, subsequently the object after separation is introduced in mass spectrum and carries out quantitative inspection
Survey；
3. carry out the quantitation of object using mass spectrum: described scanning of the mass spectrum mode is multiple-reaction monitoring, described mass spectrum is examined
Survey relevant parameter, ion source temperature is set as 550 DEG C, and spray voltage is set as 5.5kv, ion source gas 1 are 60psi, ion
Source gas 2 is 40psi, and gas curtain gas is 35psi, and the quota ion pair of 8-OhdG is m/z 284.1 > 168.0, qualitative
Ion pair is m/z 284.1 > 117.0, Isotopic Internal Standard [15n5] 8-ohdg quota ion pair m/z 289.1 > 173.0, collision
Energy is respectively 18v, 22v and 18v, and solution cluster voltage is 45v；Residence time is 150ms.
By preparing the standard substance of 1nm-300nm scope variable concentrations, draw standard curve, obtaining linear equation is y=
0.0278x+0.0515, described x represent the concentration of 8-OhdG, described y represent 8-ohdg/ [15n5]8-ohdg
Mass spectrum peak area ratio.8-OhdG concentration in urine is calculated by the mass spectrum peak area recording.
8-ohdg is a kind of endogenic mutagenic agent, has mutation tendency, leads to base mispairing in dna reproduction process, lure
Send out dna mutation, be a kind of reaction mutagenesis and the good label of canceration danger.It is as endogenouss and extrinsic factor pair
The biomarker of dna oxidative injury, is an extremely promising index.Can be assessed in vivo by the detection of 8-ohdg
The degree of oxidative damage, the mutual relation that oxidative stress is damaged with dna.8-ohdg stable existence in vivo, is that the end of metabolism produces
Thing, will not be reused by organism.And the comparision contents of 8-ohdg are stable in urine, it is not subject to diet, cell turnover etc. outer
The impact of boundary's condition.
The detection method of the present invention is applied to early stage colorectal cancer information early warning, detects from the urine sample of testee
8-OhdG, described 8-OhdG carries out pre-treatment by Solid-Phase Extraction, subsequently uses liquid chromatography mass
Multiple techniques carries out detection by quantitative.Colorectal cancer risk prediction is set up according to urine 8-OhdG concentration and age
Model carries out colorectal cancer risk analysis to testee, thus realizing the early screening of colorectal cancer damage-free type.Morbidity
Probability calculation formula:
When urine 8-OhdG concentration is more than or equal to or is less than 1.5nmol/mmol creatinine, grade of
Urinary8-ohdg value is respectively 1 and 0.When the probit calculating is more than 0.5, show that its colorectal cancer risk is high
It is less than 0.5 crowd in probit.Establish logistic regression model and carried out roc tracing analysiss, under the curve obtaining
Area value is 0.853, shows that this model has good prediction effect to the onset risk of colorectal cancer.
The detection method that the present invention provides, the collection of its specimen has non-invasive, obtains easily, the advantages of amount to obtain is many,
And it is more easy to detected person's acceptance compared to fobt test, the mental burden of staff not only can be reduced, also can allow commonly big
Crowd is more easily accepted by, and combines the method for Ultra Performance Liquid Chromatography tandem mass spectrum come accurate quantification using the offline Solid-Phase Extraction art optimizing
Dna oxidative injury markers 8-OhdG (8-ohdg) concentration in testee's urine, by the Colon and rectum set up
Cancer morbidity forecast model carries out onset risk analysis to testee.Early discovery for colorectal cancer is had for one with diagnosis offer
The new examination detection meanss of meaning.The present invention is according to the inspection of object 8-OhdG concentration in testee's urine
Survey, thus to predict the onset risk of colorectal cancer.
The present invention carries out pre-treatment with solid phase extraction techniques to urine specimen, subsequently uses Ultra Performance Liquid Chromatography technology
Urine specimen is carried out separate, then proceed to carry out detection by quantitative in the linear ion hydrazine triple level Four bar mass spectrums of series connection.The present invention
Using urine 8-OhdG as a kind of damage-free type liquid mark, it is successfully applied in the early screening of colorectal cancer.
Compared with existing colorectal cancer mark and its detection method, the present invention has the advantage that
1. the detection specimen of the present invention is urine, with respect to other specimen such as blood, saliva, sputum and feces, its specimen
Collection easy, collecting amount is big, is especially suitable for clinical research and the practice of great amount of samples；Compared to its collection of blood sample no
Wound；Compared to enteroscopy, the masses are more easily accepted by, and degree of cooperation is higher, and the smooth development benefit for screening is bigger；Phase
Compared with sputum, stool sample its be more easy on biological character as checking staff's physiology and psychologically being accepted, more people
Civilian feelings.
2., compared with conventional blood markers thing, the 8-OhdG stable existence in vivo of urine detection, is generation
The end-product thanked, and can only be formed by dna oxidative damage, therefore its detection by quantitative more can accurately reflect the morbidity of colorectal cancer
Risk.
3. blood, other humoral specimen such as feces is easily affected by external conditions such as diet, false positive rate and false negative rate
Higher, and urine 8-OhdG then avoids this difficult point well, it is not affected by diet etc., therefore detection kidney-Yang
Property rate high, false positive rate is low.
4., compared with conventional detection method, the method for the Ultra Performance Liquid Chromatography tandem mass spectrum that the present invention provides is detecting urine
Mark in liquid, it has very high sensitivity, and measurement micromolecular compound degree of accuracy is high with accuracy, and analysis time is short,
Flux is high, method favorable reproducibility.
Brief description
Fig. 1: Mobile Phase Additives acetic acid, formic acid, ammonium hydrogen carbonate are to the Mass Spectrometer Method sensitivity of 8-OhdG
Impact (mobile phase includes mobile phase a and mobile phase b, and b is pure ethanol, and Gradient elution pattern is 92.5%a and 7.5%b,
Speed is 0.25ml/min, and 8-OhdG concentration is 20nm, and sample size is 5.0ul).
Fig. 2: Mobile Phase Additives ammonium formate, the ammonium formate/formic acid of ph 3.5, the ammonium formate/ammonia of ph 9.5 are to 8- hydroxyl
(mobile phase includes mobile phase a and mobile phase b, and b is pure ethanol, waits ladder for the impact of the Mass Spectrometer Method sensitivity of base deoxyguanosine
Degree elution mode is 92.5%a and 7.5%b, and speed is 0.25ml/min, and 8-OhdG concentration is 20nm, sample size
For 5.0ul).
Fig. 3: Mobile Phase Additives ammonium acetate, the ammonium acetate/acetic acid of ph 5.0, the ammonium acetate/ammonia of ph 9.0 are to 8- hydroxyl
(mobile phase includes mobile phase a and mobile phase b, and b is pure ethanol, waits ladder for the impact of the Mass Spectrometer Method sensitivity of base deoxyguanosine
Degree elution mode is 92.5%a and 7.5%b, and speed is 0.25ml/min, and 8-OhdG concentration is 20nm, sample size
For 5.0ul).
Fig. 4: Ultra Performance Liquid Chromatography tandem mass spectrometry identifies the 8-OhdG in urine specimen.
Fig. 5: the quantitation of 8-OhdG and statistical analysis in human urine sample；Wherein a:142 example health aspiration
The content of 8-OhdG in the urine of person and 84 PATIENTS WITH LARGE BOWEL, b: in the urine of colorectal cancer i to iv phase patient
The content of 8-OhdG, c: colorectal cancer i, ii phase does not occur the patient shifting and iii, iv phase that the Urinary of transfer occurs
The content of 8-OhdG in liquid, d: the roc tracing analysiss of 8-OhdG in urine.
Specific embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments.
Embodiment 1
In the present invention, the humoral specimen of the 8-OhdG for measuring providing is urine.
A. described in step a for measure before urine sample pretreatment, first by urine under 4 degrees Celsius with 13000r/
Take supernatant 0.5ml after min centrifugation 15min, be diluted with water to 1ml, described diluent adds Isotopic Internal Standard 10pmol.
B. it is that Solid-Phase Extraction pre-treatment is carried out to urine described in step b.Described Solid-Phase Extraction is to hlb oasis post
Add 1.0ml methanol to be activated in son, add 1.0ml water and be balanced, add urine specimen.Subsequently first use
1.0ml5% methanol is carried out, and finally carries out eluting with 1ml 50% methanol.Collect eluent and be dried with vacuum centrifuge,
Subsequently add the redissolution of 0.2ml water to be measured.
C. it is to carry out separating by urine application ultra-performance liquid chromatography to be measured after pre-treatment described in step c.The present invention
In Ultra Performance Liquid Chromatography separation method chromatographic column be waters beh c18 post (1.7 μm, 2.1 × 100mm)；Column temperature is
40℃；Mobile phase is 0.1% acetic acid aqueous solution-methanol (92.5:7.5, v/v)；Flow velocity is 0.25ml/min；Applied sample amount is micro- for 5
Rise.
Wherein 0.1% acetic acid is conducive to [8-ohdg+h] during electron spray ionisation+The formation of ion, suppresses 8-ohdg
Form complex with metal ion.Applicant has also selected other solution such as 0.1% formic acid；2mm ammonium hydrogen carbonate；2mm ammonium formate；
2mm ammonium formate/formic acid, ph 3.5；2mm ammonium formate/ammonia, ph 9.5；2mm ammonium acetate；2mm ammonium acetate/acetic acid, ph5；2mm
Ammonium acetate/ammonia, ph 9, result shows, during electron spray ionisation, these mobile phases can promote [8-ohdg+na]+[8-
ohdg+k]+The formation of ion, and inhibit [8-ohdg+h]+The generation of ion.Therefore, the mobile phase 0.1% that the present invention uses
Acetic acid aqueous solution-methanol (92.5:7.5, v/v), can make the signal response intensity of 8-OhdG improve 2.7-5.3
Times.
D. it is the object isolated in step c to be introduced in mass spectrum detected described in step e, matter in the present invention
Spectrum detection method is multiple-reaction monitoring, and described Mass Spectrometer Method relevant parameter is: ion source temperature is set as 550 DEG C, spraying electricity
Pressure is set as 5.5kv, and ion source gas 1 are 60psi, and ion source gas 2 are 40psi, and gas curtain gas is 35psi.8- hydroxyl deoxidation
The quota ion pair of guanosine is m/z 284.1 > 168.0, and qualitative ion pair is m/z 284.1 > 117.0, Isotopic Internal Standard [15n5]
Quota ion pair m/z 289.1 of 8-ohdg > 173.0, collision energy is respectively 18v, 22v and 18v, and solution cluster voltage is 45v；Stay
The time is stayed to be 150ms.
E. 8-OhdG concentration in urine is calculated by the last mass spectrum peak area obtaining of step e, standard is bent
Line linear equation is y=0.0278x+0.0515, and described x represents the concentration of 8-OhdG, and described y represents 8-
ohdg/[15n5] 8-ohdg mass spectrum peak area ratio.The concentration value obtaining is compared with threshold value 1.5nmol/mmol creatinine level,
Be considered that the risk that it suffers from colorectal cancer is less than the testee of threshold level higher than the testee of threshold level 3.68
Times.Meanwhile, incidence rate is calculated with the age according to 8-OhdG concentration value, testee's quilt that probit is more than 0.5
It is considered that there is higher colorectal cancer risk.Probability calculation formula is:
In the present invention, threshold level be set as that 84 colorectal cancer patients do not occur the normal of colorectal cancer with 142
The statistical analysiss result of the 8-OhdG concentration value measured by individuality.
F. Ultra Performance Liquid Chromatography mass spectrometric hyphenated technique Methodological evaluation: precision be by measurement in a few days with difference in the daytime
Relative standard deviation assessing, described withinday precision is 1.0% to 1.9%, and described day to day precision is 0.9%
To 1.4%.Accuracy is 98.0% to 101.7%, all within the acceptable range.Matrix effect is investigated by slope ratio,
It is 91.5%, and described slope value shows that the impact of matrix effect is negligible.
G. the industrial applicability of the present invention: according to the method for examination colorectal cancer provided by the present invention, can be by urine
The risk that in liquid, the concentration of the 8-OhdG person that comes predicted detection suffers from colorectal cancer, can take correlation means to carry out according to this
Early intervention, thus stoping the progress of colorectal cancer, improves China's heath economics level, therefore this invention is effective.
Embodiment 2
Test example 1 (applies solid phase extraction techniques, Ultra Performance Liquid Chromatography tandem mass spectrometry to detect 8- in normal human urine
Hydroxy-Guanine content)
Application to detect from the urine of 142 healthy volunteers at the attached second hospital's international health center of Zhejiang University
The content of object 8-OhdG.
Detection urine carries out pretreatment before the assay, urine is centrifuged after 15min with 13000r/min under 4 degrees Celsius and takes
Supernatant 0.5ml, is diluted with water to 1.0ml, described diluent adds Isotopic Internal Standard 10pmol, and adds 1.0ml backward
Methanol carries out the urine sample after activating injection 1.0ml dilution in the hlb oasis pillar after being balanced with 1.0ml water, on
First it is carried out with 1.0ml 5% methanol after sample, finally carries out eluting with 1.0ml 50% methanol, collect eluent and use vacuum
Centrifuge is dried, and subsequently adds 0.2ml water and redissolves.
Redissolve urine application ultra-performance liquid chromatography to be measured to carry out separating, chromatographic column is waters beh c18 post
(1.7μm,2.1×100mm)；Column temperature is 40 DEG C；Mobile phase is 0.1% acetic acid aqueous solution-methanol (92.5:7.5, v/v)；Flow velocity
For 0.25ml/min；Applied sample amount is 5 microlitres；
As shown in Figure 1, 2, 3, nine kinds of different flow visualizing, are used acetic acid can improve 8- hydroxyl as additive and take off
2.7-5.3 times of the mass spectrum response intensity of oxygen guanosine.
Finally the object isolated is introduced in mass spectrum and is detected, source temperature is set as 550 DEG C, and spray voltage sets
It is set to 5.5kv, ion source gas 1 are 60psi, ion source gas 2 are 40psi, gas curtain gas is 35psi.8-OhdG
Quota ion pair be m/z 284.1 > 168.0, qualitative ion pair be m/z 284.1 > 117.0, Isotopic Internal Standard [15n5]8-
Quota ion pair m/z 289.1 of ohdg > 173.0, collision energy is respectively 18v, 22v and 18v, and solution cluster voltage is 45v；Resident
Time is 150ms (shown in Fig. 4).
Recording the 8-OhdG measured value range in 142 healthy volunteer's urines is 0.24-2.47nmol/
Mmol creatinine, meansigma methodss are 1.07 ± 0.49nmol/mmol creatinine.
Test example 2 (applies solid phase extraction techniques, Ultra Performance Liquid Chromatography tandem mass spectrometry to detect colorectal cancer patients urine
8-OhdG content in liquid)
With the method same with test example 1, it is applied in the urine detection of colorectal cancer patients, measure 8- hydroxyl in its urine
The content of base deoxyguanosine.Application comes from the urine of 84 colorectal cancer patients of the attached second hospital's surgical oncology of Zhejiang University
Liquid specimen.
8-OhdG measured value range in the urine of 84 colorectal cancer patients is 0.51-4.37nmol/
Mmol creatinine, meansigma methodss are 1.68 ± 0.85nmol/mmol creatinine.
According to Fig. 5 a, in the urine of colorectal cancer patients, the concentration of 8-OhdG is higher than normal person, and has
Significant difference, statistically significant.This shows, by applying solid phase extraction techniques joint Liquid Chromatography-Tandem Mass Spectrometry detection urine
The concentration method of 8-OhdG in liquid, colorectal cancer is carried out with examination and early diagnosiss is possible.
As shown in Figure 5 b, from i to the iv phase, the concentration of the 8-OhdG in urine is gradually increased colorectal cancer, dna
Oxidative damage be in enhancing trend, the 8-OhdG measured value range in colorectal cancer i phase Urine in Patients be 0.68-
2.58nmol/mmol creatinine, meansigma methodss are 1.30 ± 0.61nmol/mmol creatinine；8- in colorectal cancer ii phase Urine in Patients
Hydroxy-Guanine measured value range is 0.51-3.66nmol/mmol creatinine, and meansigma methodss are 1.51 ± 0.78nmol/mmol flesh
Acid anhydride；8-OhdG measured value range in colorectal cancer iii phase Urine in Patients is 0.57-4.10nmol/mmol creatinine,
Meansigma methodss are 1.78 ± 0.84nmol/mmol creatinine；8-OhdG measured value in colorectal cancer iv phase Urine in Patients
Scope is 0.84-4.37nmol/mmol creatinine, and meansigma methodss are 2.29 ± 1.07nmol/mmol creatinine.
As shown in Figure 5 c, there is 8-OhdG in the Urine in Patients shifting in iii, iv phase colorectal cancer tumor
Concentration will be significantly higher than the patient that i, ii phase do not occur to shift, and has significant difference；Show by 8-OhdG
Concentration can assess whether colorectal cancer occurs lymphatic metastasiss, metastasis such as hepatic metastasis.
As fig 5d, carry out roc tracing analysiss, verify the power of test of the colorectal cancer of this detection method, auc more connects
Nearly 1 more display power of test is high.In the case that application the inventive method detects the concentration of 8-OhdG, auc is
0.853,95% credibility interval is 0.801-0.905, shows that its Detection results is preferable.
Table one. the logistic linear regression analyses of several risks and assumptions related to Colorectal Cancer
According to table one, the concentration of the onset risk of colorectal cancer and urine 8-OhdG, age and sex are entered
Row logistic linear regression analyses, analysis shows, in urine, 8-OhdG concentration is higher, and age bigger individuality is more
It is susceptible to suffer from colorectal cancer, and the onset risk of sex and colorectal cancer non-correlation；Additionally, being taken off according to statistical analysis 8- hydroxyl
The risk that oxygen guanosine concentration suffers from colorectal cancer higher than 1.5nmol/mmol creatinine person is less than 1.5nmol/mmol creatinine person's
3.68 times, 95% credibility interval is 1.82-7.45.
Additionally, establishing to predict Risk of Colorectal Cancer with the model of urine 8-OhdG concentration and age, and
Colorectal Cancer probability is calculated according to formula.Formula, as described above, cutoff value is 0.5, shows that the prediction of colorectal cancer is general
The risk suffering from colorectal cancer higher than 0.5 for rate value p will be significantly higher than the individuality that prediction probability value is less than 0.5.
According to above it was therefore concluded that 8-OhdG can carry out suffering from Colon and rectum as a new mark
The prediction of cancer and risk assessment.
The industrial applicability of the present invention, according to the method for examination colorectal cancer provided by the present invention, can pass through urine
The concentration of middle 8-OhdG comes predicted detection, and person suffers from the risk of colorectal cancer, and correlation means can be taken according to this to carry out morning
Phase intervenes, thus stoping the progress of colorectal cancer, improves China's heath economics level, therefore this invention is effective.
The present invention is the method for carrying out Early cancer examination to testee, applies Ultra Performance Liquid Chromatography mass spectrum
Associated with method to measure the 8-OhdG in urine, substitute into building based on urine 8-ohdg concentration and age of foundation
The incidence rate computing formula of vertical Colorectal Cancer forecast model, assesses the onset risk of its colorectal cancer.Specifically, institute
The method of stating comprises: application Solid-Phase Extraction carries out pre-treatment to urine specimen, realizes desalination, concentration and enrichment.(step a, b), with
(step c), with linear ion hydrazine series connection three to isolate the step of the 8-OhdG in urine for Ultra Performance Liquid Chromatography
Weight level Four bar mass spectrum carrys out the step (step d, e) of the content of 8-OhdG in quantitative urine, and general with morbidity
Rate computing formula is calculating colorectal cancer risk, in addition, containing the disease that detection colorectal cancer occurs in this specification
Progress extent.
Present invention research confirms, adds acetic acid in the step of liquid phase separation in mobile phase a, can improve 8- hydroxyl and take off
The mass spectrum response of oxygen guanosine.Additionally, by adding Isotopic Internal Standard, thus accurately carrying out to 8-OhdG in urine
Quantitative.In colorectal cancer patients urine, the content of 8-OhdG is higher than normal person, there is significant difference.In i
To in the colorectal cancer patients urine of iv phase, the concentration of 8-OhdG also presents the trend of rising, shows with swollen
The development of tumor, it is in serious trend that dna damages.There is 8- hydroxyl deoxidation in patient's (the iii phase and iv phase) urine shifting in tumor
The concentration of guanosine is higher than not shift patient's (i phase and ii phase), and there is significant difference.By being connected by Ultra Performance Liquid Chromatography
The determination techniques that mass spectrum is constituted, can detect to testee's urine 8-OhdG concentration, in conjunction with logistic
Analysis of regression model can be predicted to colorectal cancer risk, thus completing the present invention.
Claims (2)
1. a kind of 8-OhdG is as the application of urine detection mark, the molecule of described 8-OhdG
Formula: c10h13o5n5It is characterised in that being realized by following steps:
(1) sample is through Solid-Phase Extraction pretreatment: urine specimen to be measured is stored in -80 DEG C of refrigerators, takes out in 4 DEG C before application
Lower defrosting, takes supernatant dilute after centrifugation, and add 10pmol Isotopic Internal Standard [15n5] 8-ohdg, make Solid-Phase Extraction, to
Add methanol to be activated in hlb oasis pillar, add water and be balanced, add urine specimen, subsequently use 5% methanol
It is carried out, finally carries out eluting with 50% methanol, collect eluent and be dried with vacuum centrifuge, the redissolution that subsequently adds water is treated
Survey；
(2) urine after Solid-Phase Extraction process carries out separating using liquid chromatograph, and chromatographic condition used is: chromatographic column is
Waters beh c18 post, 1.7 μm, 2.1 × 100mm, column temperature is 40 DEG C, and mobile phase is 0.1% acetic acid aqueous solution-methanol,
92.5:7.5, v/v, flow velocity is 0.25ml/min, and applied sample amount is 5 microlitres, subsequently is introduced in mass spectrum by the object after separation
Row detection by quantitative；
(3) carry out the quantitation of object with mass spectrum, described scanning of the mass spectrum mode is multiple-reaction monitoring, described Mass Spectrometer Method is related
Parameter, ion source temperature is set as 550 DEG C, and spray voltage is set as 5.5kv, and ion source gas 1 are 60psi, ion source gas 2
For 40psi, gas curtain gas is 35psi, and the quota ion pair of 8-OhdG is m/z 284.1 > 168.0, qualitative ion pair
For m/z 284.1 > 117.0, Isotopic Internal Standard [15n5] 8-ohdg quota ion pair m/z 289.1 > 173.0, collision energy divides
Not Wei 18v, 22v and 18v, solution cluster voltage be 45v；Residence time is 150ms, by preparing standard substance, draws standard curve, leads to
Cross the mass spectrum peak area recording to calculate 8-OhdG concentration in urine.
2. as the application of urine detection mark, its feature exists a kind of 8-OhdG according to claim 1
In step (3) is passed through to prepare the standard substance of 1nm-300nm scope variable concentrations, draws standard curve, and obtaining linear equation is y
=0.0278x+0.0515, described x represent the concentration of 8-OhdG, described y represent 8-ohdg/ [15n5]8-
Ohdg mass spectrum peak area ratio, calculates 8-OhdG concentration in urine by the mass spectrum peak area recording.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610724501.3A CN106370744A (en) | 2016-08-25 | 2016-08-25 | Application of 8-hydroxydeoxyguanosine as urine marker |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108732256A (en) * | 2017-04-21 | 2018-11-02 | 凯熙医药（武汉）股份有限公司 | 8-OhdG detection kit and its application |
| CN108872563A (en) * | 2018-08-16 | 2018-11-23 | 广东省计划生育科学技术研究所（广东省计划生育专科医院） | The method of DNA fragment with spermatozoon rate, active oxygen and the high infertility of 8-OhdG association evaluation male |
| CN112198239A (en) * | 2020-08-26 | 2021-01-08 | 深圳康谱生物科技有限公司 | Method for rapidly detecting 8-OHDG in urine by combining UPLC-MS/MS technology |
| CN113777209A (en) * | 2021-11-08 | 2021-12-10 | 生态环境部华南环境科学研究所 | Synchronous detection and application of exposure and effect markers of volatile pollutants in urine |
| CN114354807A (en) * | 2021-12-31 | 2022-04-15 | 天津诺禾医学检验所有限公司 | Method for detecting 8-hydroxy-2-deoxyguanosine |
| CN114755422A (en) * | 2022-06-10 | 2022-07-15 | 杭州凯莱谱精准医疗检测技术有限公司 | Biomarker for colorectal cancer detection and application thereof |
| CN115487542A (en) * | 2022-09-02 | 2022-12-20 | 浙江大学 | Solid-phase extraction method for modified nucleosides in urine and application of solid-phase extraction method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104458988A (en) * | 2014-11-27 | 2015-03-25 | 山东出入境检验检疫局检验检疫技术中心 | Method for testing group of biomarkers |
-
2016
- 2016-08-25 CN CN201610724501.3A patent/CN106370744A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104458988A (en) * | 2014-11-27 | 2015-03-25 | 山东出入境检验检疫局检验检疫技术中心 | Method for testing group of biomarkers |
Non-Patent Citations (2)
| Title |
|---|
| LILY L. WU ET AL: "Urinary 8-OHdG: a marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics", 《CLINICA CHIMICA ACTA》 * |
| 郭成 等: "同位素稀释质谱法研究 DNA氧化损伤与大肠癌发生发展关联", 《中国化学会第二届全国质谱分析学术报告会会议摘要集》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108732256A (en) * | 2017-04-21 | 2018-11-02 | 凯熙医药（武汉）股份有限公司 | 8-OhdG detection kit and its application |
| CN108872563A (en) * | 2018-08-16 | 2018-11-23 | 广东省计划生育科学技术研究所（广东省计划生育专科医院） | The method of DNA fragment with spermatozoon rate, active oxygen and the high infertility of 8-OhdG association evaluation male |
| CN112198239A (en) * | 2020-08-26 | 2021-01-08 | 深圳康谱生物科技有限公司 | Method for rapidly detecting 8-OHDG in urine by combining UPLC-MS/MS technology |
| CN113777209A (en) * | 2021-11-08 | 2021-12-10 | 生态环境部华南环境科学研究所 | Synchronous detection and application of exposure and effect markers of volatile pollutants in urine |
| CN113777209B (en) * | 2021-11-08 | 2022-02-08 | 生态环境部华南环境科学研究所 | Synchronous detection and application of exposure and effect markers of volatile pollutants in urine |
| CN114354807A (en) * | 2021-12-31 | 2022-04-15 | 天津诺禾医学检验所有限公司 | Method for detecting 8-hydroxy-2-deoxyguanosine |
| CN114755422A (en) * | 2022-06-10 | 2022-07-15 | 杭州凯莱谱精准医疗检测技术有限公司 | Biomarker for colorectal cancer detection and application thereof |
| CN114755422B (en) * | 2022-06-10 | 2022-10-21 | 杭州凯莱谱精准医疗检测技术有限公司 | Biomarker for colorectal cancer detection and application thereof |
| CN115487542A (en) * | 2022-09-02 | 2022-12-20 | 浙江大学 | Solid-phase extraction method for modified nucleosides in urine and application of solid-phase extraction method |
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