CN109917042A - A kind of kit - Google Patents
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- CN109917042A CN109917042A CN201910274765.7A CN201910274765A CN109917042A CN 109917042 A CN109917042 A CN 109917042A CN 201910274765 A CN201910274765 A CN 201910274765A CN 109917042 A CN109917042 A CN 109917042A
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Abstract
The present invention provides a kind of kit suitable for assessing the postoperative late recurrent risk of atrial fibrillation catheter ablation.And this kit is the kit for measuring plasma TM AO content, by being measured to TMAO in patients blood plasma, the postoperative risk of recurrence of patients with atrial fibrillation catheter ablation can effectively be assessed, patient catheter can be assessed in the preoperative convenient for doctor melts late recurrence after surgery risk, and then specified targeted treatment schemes, it provides for patient and timely and effectively treats.
Description
Technical field
The present invention relates to field of medical technology more particularly to a kind of kits.
Background technique
Atrial fibrillation is that clinically one of most common arrhythmia cordis, disease incidence and illness rate increase with the age and gradually increase
Add.Cerebral apoplexy, heart failure, the sudden cardiac arrest etc. that atrial fibrillation causes can seriously increase lethality, disability rate, bring heavy medical treatment
Burden.There are many kinds of the treatment means for treating atrial fibrillation, and catheter ablation is wherein important one kind.However atrial fibrillation catheter ablation
Late recurrent rate afterwards reaches 25%-40%, therefore assesses the postoperative risk of recurrence of atrial fibrillation catheter ablation in the preoperative and specific aim is specified to control
Treatment scheme is of great significance.
The pathophysiological mechanism of atrial fibrillation not yet illustrate completely it is clear, typically now think atrial fibrillation be by initiating triggering because
What element and maintenance matrix interaction generated.When autonomic nerve stimulation, ischemic, great cardiac vein (including pulmonary vein, chamber
Vein etc.) flesh sleeve in Abnormal automaticity cell can spontaneous generation rapid electrical activity, such abnormal electrical activity is the beginning of atrial fibrillation
Dynamic triggering factors.The abnormal electrical activity of initiating triggering propagates to heart muscle and causes multiple wavelets of turning back, so as to cause the shape of atrial fibrillation
At.Atrial fibrillation once trigger formed can self―sustaining, and this maintain i.e. dependent on atrium electricity reconstruct and structural remodeling.Electricity reconstruct
Basis be that atrial muscle cell gene expression can change when atrial fibrillation, atrial stretch, and then cause cell surface
Ion channel changes.The variation of myocardial cell surface ion channel and then the variation for causing transmembrane currents, as L-type calcium is logical
The reduction of road ion stream, transient outward K+, Delayed Rectifier Potassium Current, inward rectifyimg potassium channel and ATP sensitivity potassium are logical
The increase of road electric current finally causes the effective refractory period of atrial muscle cell to shorten, inhomogeneity increases, frequency adaptability is bad.
The lasting structural remodeling for also resulting in atrium of atrial fibrillation.The structural remodeling that atrial fibrillation causes includes Gap Junction Remodelling, thin
Born of the same parents' reconstruct.Gap Junction Remodelling shows as the expression and distribution point heterogeneity of connection albumen；Cell reconstitution is withered by cardiac muscle cell
It dies, the Atrial fibrosis that myolysis and cardiac interstitium fibroplasia cause.The change of atrial muscle cell ultra microstructure, cardiac muscle fibre
Change, the redistribution of connection albumen make excitement pass to slow down, tortuous path, the final generation and maintenance for promoting atrial fibrillation.
Research finds that atrial fibrillation itself can cause atrial electrical remodeling and structural remodeling, and then shows as " atrial fibrillation to atrial fibrillation " effect
It answers, and the breaking-out of atrial fibrillation can seriously increase lethality, disability rate, therefore the sinus rhythm for restoring patients with atrial fibrillation is significant.Its
In, catheter ablation makes to restore one of the important means of patients with atrial fibrillation sinus rhythm.In the electrophysiologic study of Patients with Paroxysmal Atrial Fibrillation
Acceptance of the bid measures 90% or more triggering stove and is respectively positioned on inside pulmonary vein, thus pulmonary vein electric isolution be current atrial fibrillation ablation it is most basic,
The most frequently used art formula.However, early postoperation (in March) recurrence rate is up to 50% or more, the fortunately atrial fibrillation of early stage recurrence after surgery 3
It is interior to die away, therefore at this stage using after ablation 3 months as the judgement time reference evaluated whether melting non-late recurrent.
Late Stage of Postoperative (in December) recurrence rate reaches 25%-40%, and the probability recurred for the first time after melting 1 year researches show that art is few, therefore
It is most commonly used within postoperative 1 year at this stage the time of the evaluation and test postoperative recurrence of atrial fibrillation catheter ablation.Researches show that late recurrence after surgery with
The degree of Remodelling in Atrial Fibrillation has correlation, and mapping shows that the mechanism of conduit late recurrence after surgery is pulmonary vein in electrophysiologic study
The triggering of the logical again of isolation, pulmonary vein external trigger stove, and previously research shows that TMAO can pass through activation TGF β-P-SMAD3 letter
Number access and then promote myocardial fibrosis, shortens cardiac muscle cell's effective refractory period by activation NF- κ B signal access increasing, therefore
We are it is believed that TMAO may play a role in atrial fibrillation pipe ablation late recurrence after surgery.
With the continuous understanding to atrial fibrillation pathogenesis, the relevant harm of atrial fibrillation and recovery patients with atrial fibrillation sinus rhythm are gradually
It is taken seriously, and sensitive, the special biological marker still without the assessment postoperative late recurrent risk of atrial fibrillation catheter ablation at present
Object, therefore be badly in need of that our the world of medicine experts and scholars is wanted to study a kind of postoperative late recurrent sensibility of assessment atrial fibrillation catheter ablation jointly
It easily surveys, the method that specificity is high, assesses postoperative recurrence risk as early as possible to preoperative, doctor is made to carry out individualized treatment for patient
(such as compound art formula treatment on the basis of ring pulmonary vein isolation).
Summary of the invention
The purpose of the present invention is to solve disadvantages existing in the prior art, and the one kind proposed disappears for atrial fibrillation conduit
Melt the kit of the assessment of postoperative recurrence.
To achieve the goals above, present invention employs following technical solutions:
A kind of kit of the invention, including following components: acetonitrile (chromatographically pure), ammonium acetate (chromatographically pure), TMAO standard items are (pure
Degree >=99%), deuterated trimethylamine oxide (d9-TMAO) (purity >=98%)；Its kit is used to detect the efficient liquid phase of TMAO
The mass spectrographic kit of chromatographic tandem.And the kit is used for the assessment of the postoperative recurrence of atrial fibrillation catheter ablation.
The present inventor is by having done in-depth study, experiment to catheter ablation is postoperative to patients with atrial fibrillation morbidity early period
Have found a kind of kit suitable for assessing the postoperative late recurrent risk of atrial fibrillation catheter ablation.And this kit is for surveying
The kit for determining plasma TM AO content can effectively assess patients with atrial fibrillation catheter ablation by measuring to TMAO in patients blood plasma
Postoperative risk of recurrence can assess patient catheter convenient for doctor in the preoperative and melt late recurrence after surgery risk, and then specify and be directed to
Property therapeutic scheme, provides for patient and timely and effectively treats.
The assessment kit of the postoperative late recurrent of heretofore described catheter ablation is by TMAO in patients blood plasma
Measurement assess patient's postoperative recurrence risk.TMAO is choline, the lecithin and left-handed decomposed in food by enteric microorganism
Carnitine generates trimethylamine, and then the portal vein system is absorbed into Liver Channel flavin-containing mono-oxygenase and aoxidizes.TMAO enters energy after blood
Cardiovascular system is enough set to generate a variety of pathophysiological changes, such as by activating TGF β-P-SMAD3 signal path to promote cardiac muscle
Fibrosis；By activation NF- κ B signal access increase cardiac nerve ganglionic layer inflammatory factor expression make neuromere function and
Activity increases, effective refractory period shortens.The present inventor warp experimental studies have found that, pass through high performance liquid chromatography tandem mass spectrum hair measurement
The expression of the plasma TM AO of patients with atrial fibrillation finds plasma TM AO in the patients with atrial fibrillation of the postoperative recurrence of catheter ablation compared with catheter ablation
Postoperative non-late recurrent group content is significantly raised, and gap has statistical significance (it is expected that P < 0.05).Illustrate to disappear in atrial fibrillation conduit
Melt postoperative recurrence patients blood plasma's TMAO higher value of clinical studies, the postoperative recurrence of patients with atrial fibrillation catheter ablation and the close phase of TMAO
It closes.By being used as the important of the postoperative late recurrent risk assessment of catheter ablation to patients with atrial fibrillation plasma TM AO quantitative detection
Foundation and the evaluation index for formulating reasonable individualized treatment scheme.
TMAO assesses kit, that is, TMAO high performance liquid chromatography tandem mass spectrum kit, application method:
The configuration of standard items: mother liquor: ultrapure water configures the standard TMAO stock solution of 1mg/ml, and -20 DEG C of refrigerator storages are spare.Except egg
White liquor: ultrapure water configures the d9-TMAO internal standard solution of 0.7mg/ml, and with 500 times of dilution in acetonitrile, -20 DEG C of refrigerator storages are standby
With.
The configuration of standard curve: detection before TMAO mother liquor is diluted step by step, be configured to various concentration standard solution (50,
125,250,500,1250,2500,5000ng/mL), the removing protein liquid in A is added, whirlpool shakes 1min, at 4 DEG C,
13000rpm is centrifuged 15min.Using sample introduction concentration as abscissa, peak area is that ordinate draws standard curve.
The processing of sample: taking 20 μ L plasma samples, and the removing protein liquid being added in the 80 above-mentioned A of μ L is put into centrifuge tube, shakes
1min is sufficiently mixed uniformly, and 4 DEG C, 13000rpm is centrifuged 15min.Supernatant is taken to be transferred to sample injection bottle, machine testing in preparation.
The adjusting of chromatographic condition: 1502.1mm, 1.7 μm of hydrophilic chromatographic column are used；Mobile phase A: water (5mmol acetic acid
Ammonium)；Mobile phase B: acetonitrile.It is eluted using Gradient program, gradient condition: 0-5min, 50%A；5-10min, 50%A-80%, 10-
15min, 80%A run total time 15min；Flow velocity: 0.3mL/min；Sample volume: 3 μ L；Column temperature: 30C.
The adjusting of Mass Spectrometry Conditions: ion source: electric spray ion source (ESI), positive ion mode；High pure nitrogen (purity
99.999%) as dry gas, dry temperature degree 325C, dry gas stream speed 10L/min；Scanning mode: multiple-reaction monitoring pattern
(MRM), quota ion pair: TMAOm/z76 → 58, d9-TMAOm/z85 → 66；Qualitative ion: TMAOm/z42, d9-TMAOm/
z46；Collision energy: TMAO20eV, d9-TMAO25eV.
TMAO detection: being quantified using d9-TMAO as Isotopic Internal Standard, and formic acid, methanol are obtained as flowing A, B phase
Mass spectrogram, peak area are converted into chromatographic data through computer and obtain test plasma sample TMAO concentration.
Detailed description of the invention
Fig. 1 is EIC figure of the TMAO in blank solution；
Fig. 2 is EIC figure of the d9-TMAO in blank solution；
Fig. 3 is EIC figure of the TMAO in blood plasma；
Fig. 4 is EIC figure of the d9-TMAO in blood plasma；
The ROC curve of Fig. 5 TMAO and the postoperative late recurrent risk of atrial fibrillation catheter ablation.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.
Embodiment 1: the clinical research of atrial fibrillation catheter ablation postoperative late recurrent and plasma TM AO
One, case is chosen
Selection will receive catheter ablation for the first time, and art formula is patients with atrial fibrillation 60 of pulmonary vein isolation art, respectively at March, June, 9
The moon, follow-up in December atrial fibrillation recurrence situation, it is divided into non-late recurrent group and postoperative recurrence group；It is supraventricular aroused in interest to choose 30 paroxysmals
Patient 30 are overrun as control group.[exclusion criteria: previously there is atrial fibrillation ablation history；There are left atrial thrombus；There are anticoagulant taboos；Six
A month myocardium infarct history；Congestive heart failure；Serious chronic organic disease；Hyperthyroidism；The gestational period；Spirit is different
Often]
Two, the measurement result of plasma TM AO
Preoperative early morning empty stomach venous blood samples 3ml is placed in EDTA anti-coagulants vacuum tube, and 4 DEG C of 3500rpm are centrifuged l0min, separation
Blood plasma out is set -80 DEG C of refrigerators and is saved.Sample is quantitative determined using high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) respectively
Plasma TM AO.
3 groups of plasma TM AO values are shown in Table 1.TMAO recurrence group expression after surgery is apparently higher than non-late recurrent group and paroxysmal room
Upper property tachycardia control group, difference are statistically significant (P < 0.05).There is document report can be 4 etc. by plasma TM AO level point
Grade, is 1 grade respectively: < 2.43umol/L；2 grades: 2.43 ~ 3.66umol/L；3 grades: 3.67 ~ 6.18umol/L；4 grades: >
6.18umol/L.This research postoperative recurrence group is similar to above-mentioned 3 grades of levels, and successful surgery group is horizontal similar to above-mentioned 1-2 grades, right
It is similar to above-mentioned 1 grade of level according to organizing.
Table 1:3 group plasma TM AO, TGF-β and LA Tmsv-SD compare
| Grouping | Control group | Non- late recurrent group (n=41) | Postoperative recurrence group (n=19) | |
| Plasma TM AO (μm ol/L) | 2.10±0.12 | 2.14±0.49 | 4.22±0.18* | |
| TGF-β(ng/mL) | 24.62±6.11* | 27.33±9.81 | 34.63±11.98* | |
| LA Tmsv-SD | 8.1±3.63* | 12.0±4.83 | 15.5±5.57* |
Note: LA Tmsv-SD: the asynchronous index of left room.* P < 0.05 compared with non-late recurrent group.
Further to assess TMAO to the predictive value of the postoperative late recurrent of atrial fibrillation catheter ablation, with atrial fibrillation catheter ablation
Late recurrent is diagnostic criteria afterwards, by calculating the sensitivity under different TMAO levels, specificity index, draws ROC curve, obtains
Obtaining TMAO is 0.875 to area under the ROC curve of the postoperative late recurrent of atrial fibrillation catheter ablation, and display TMAO disappears to atrial fibrillation conduit
Melt late recurrence after surgery to be worth with middle Height Prediction, sees Fig. 5.
Three, conclusion
The present inventor it has been investigated that, plasma TM AO in the more non-late recurrent group of the postoperative recurrence group of atrial fibrillation catheter ablation and
Control group plasma TM AO content is high, illustrates that the content height of plasma TM AO in the atrial fibrillation recurred after surgery has value of clinical studies, room
The postoperative recurrence of the catheter ablation that quivers and plasma TM AO are in close relations.In addition, this research also found, the blood plasma of postoperative recurrence patient
The asynchronous index of TGF-β, left room is apparently higher than non-late recurrent group, and plasma TM AO and blood plasma TGF-β, left room synchronize index
It is positively correlated, this shows TMAO likely via TGF β-P-SMAD3 signal path and then promotes myocardial fibrosis, and then causes
Left room asynchronization activity, causes the postoperative late recurrent of atrial fibrillation catheter ablation.This research is analyzed while being found by ROC curve
TMAO is worth the postoperative late recurrent of atrial fibrillation catheter ablation with middle Height Prediction.
So plasma TM AO can be used as the sensitive evaluation index of the postoperative late recurrent of atrial fibrillation catheter ablation.Detect blood plasma
TMAO can be used for patients with atrial fibrillation preoperative evaluation to formulate individualized treatment scheme.
Embodiment 2: the concrete case in embodiment 1
Patient one
One, clinical data
Patient, male, 53 years old, because " palpitaition is more than half a year " is admitted to hospital, diagnosis: auricular fibrillation (paroxysmal), row ring pulmonary vein isolation art, art
Mesostroma mapping does not find left room low-voltage area.Post-operative recovery sinus rhythm.It is multiple respectively at postoperative March, June, September, December
It looks into, has no atrial fibrillation recurrence.
Two, the measurement of plasma TM AO:
TMAO high performance liquid chromatography tandem mass spectrum kit measurement method: (1) the preoperative limosis vein blood 3ml of patient is acquired, is placed in
EDTA anti-coagulants vacuum tube, 4 DEG C of 3500r are centrifuged l0min, isolate blood plasma, set -80 DEG C of refrigerators and save.(2) kit is taken out
Configuration standard product: mother liquor: ultrapure water configures the standard TMAO stock solution of 1mg/ml, and -20 DEG C of refrigerator storages are spare.Removing protein liquid:
Ultrapure water configures the d9-TMAO internal standard solution of 0.7mg/ml, and with 500 times of dilution in acetonitrile, -20 DEG C of refrigerator storages are spare.(3)
Configuration standard curve: TMAO mother liquor is diluted step by step, be configured to various concentration standard solution (50,125,250,500,1250,
2500,5000ng/mL), removing protein liquid is added, whirlpool shakes 1min, and at 4 DEG C, 13000rpm is centrifuged 15min.Take supernatant into
Sample detection is drawn using sample introduction concentration as abscissa, and peak area is that ordinate draws standard curve.(4) patient's blood specimen is taken out,
It is placed in thaw at RT.20 μ L plasma samples are taken, 80 μ L removing protein liquid are added and are put into centrifuge tube, concussion 1min is sufficiently mixed
Even, 4 DEG C, 13000rpm is centrifuged 15min.Supernatant is taken to be transferred to sample injection bottle, machine testing in preparation.(5) it adjusts chromatographic condition: using
1502.1mm, 1.7 μm of hydrophilic chromatographic column；Mobile phase A: water (5mmol ammonium acetate)；Mobile phase B: acetonitrile.Using Gradient program
Elution, gradient condition: 0-5min, 50%A；5-10min, 50%A-80%, 10-15min, 80%A run total time 15min；Stream
Speed: 0.3mL/min；Sample volume: 3 μ L；Column temperature: 30 DEG C.(6) Mass Spectrometry Conditions: ion source: electric spray ion source (ESI) are adjusted, just
Ion mode；High pure nitrogen (purity 99.999%) is as dry gas, dry 325 DEG C of temperature degree, dry gas stream speed 10L/min；It sweeps
Retouch mode: multiple-reaction monitoring pattern (MRM), quota ion pair: TMAOm/z76 → 58, d9-TMAOm/z85 → 66；It is qualitative from
Son: TMAOm/z42, d9-TMAOm/z46；Collision energy: TMAO20eV, d9-TMAO25eV.(7) TMAO is detected: with d9-TMAO
It is quantified as Isotopic Internal Standard, by flowing A, B phase, obtains mass spectrogram, peak area is converted into chromatographic data through computer
Obtain patients blood plasma's sample TMAO concentration.As a result as shown in Figure 3 as a result, going out the preoperative sky of the patients with atrial fibrillation using this kits
Abdomen plasma TM AO content is 2.2 μm of ol/L.
Three, conclusion
The preoperative plasma TM AO content of patient is that 2.2 μm of ol/L(assume), it is similar to 1 grade of level of plasma TM AO in embodiment 1,
Late recurrent rate after predicting patient catheter's ablation is low.The postoperative periodic review for having carried out 1 year by a definite date of patient does not have atrial fibrillation multiple
Hair.Therefore, the preoperative low-level plasma TM AO assessment late recurrence after surgery risk of the patient is low and expected consistent.
Patient two
One, clinical data
Patient, female 67 years old, because " pareordia discomfort is more than January repeatedly " is admitted to hospital, diagnose: auricular fibrillation (paroxysmal), row ring pulmonary vein
Sequestration, art mesostroma mapping are shown in left posterior wall sheet low-voltage area, and low-voltage area is isolated for parallel local host improvement.Art
After restore sinus rhythm.Respectively at postoperative March, June, September, check in December, Yu Shuhou September check discovery atrial fibrillation is sent out again.
Two, the measurement of plasma TM AO:
TMAO high performance liquid chromatography tandem mass spectrum kit measurement method: (1) the preoperative limosis vein blood 3ml of patient is acquired, is placed in
EDTA anti-coagulants vacuum tube, 4 DEG C of 3500r are centrifuged l0min, isolate blood plasma, set -80 DEG C of refrigerators and save.(2) kit is taken out
Configuration standard product: mother liquor: ultrapure water configures the standard TMAO stock solution of 1mg/ml, and -20 DEG C of refrigerator storages are spare.Removing protein liquid:
Ultrapure water configures the d9-TMAO internal standard solution of 0.7mg/ml, and with 500 times of dilution in acetonitrile, -20 DEG C of refrigerator storages are spare.(3)
Configuration standard curve: TMAO mother liquor is diluted step by step, be configured to various concentration standard solution (50,125,250,500,1250,
2500,5000ng/mL), removing protein liquid is added, whirlpool shakes 1min, and at 4 DEG C, 13000rpm is centrifuged 15min.Take supernatant into
Sample detection is drawn using sample introduction concentration as abscissa, and peak area is that ordinate draws standard curve.(4) patient's blood specimen is taken out,
It is placed in thaw at RT.20 μ L plasma samples are taken, 80 μ L removing protein liquid are added and are put into centrifuge tube, concussion 1min is sufficiently mixed
Even, 4 DEG C, 13000rpm is centrifuged 15min.Supernatant is taken to be transferred to sample injection bottle, machine testing in preparation.(5) it adjusts chromatographic condition: using
1502.1mm, 1.7 μm of hydrophilic chromatographic column；Mobile phase A: water (5mmol ammonium acetate)；Mobile phase B: acetonitrile.Using Gradient program
Elution, gradient condition: 0-5min, 50%A；5-10min, 50%A-80%, 10-15min, 80%A run total time 15min；Stream
Speed: 0.3mL/min；Sample volume: 3 μ L；Column temperature: 30 DEG C.(6) Mass Spectrometry Conditions: ion source: electric spray ion source (ESI) are adjusted, just
Ion mode；High pure nitrogen (purity 99.999%) is as dry gas, dry 325 DEG C of temperature degree, dry gas stream speed 10L/min；It sweeps
Retouch mode: multiple-reaction monitoring pattern (MRM), quota ion pair: TMAOm/z76 → 58, d9-TMAOm/z85 → 66；It is qualitative from
Son: TMAOm/z42, d9-TMAOm/z46；Collision energy: TMAO20eV, d9-TMAO25eV.(7) TMAO is detected: with d9-TMAO
It is quantified as Isotopic Internal Standard, by flowing A, B phase, obtains mass spectrogram, peak area is converted into chromatographic data through computer
Obtain patients blood plasma's sample TMAO concentration.As a result as shown in Figure 3 as a result, going out the preoperative sky of the patients with atrial fibrillation using this kits
Abdomen plasma TM AO content is 4.19 μm of ol/L.
Three, conclusion
The preoperative plasma TM AO content of patient is 4.19 μm of ol/L, similar to 3 grades of levels of plasma TM AO in embodiment 1, and prediction is suffered from
The postoperative late recurrent rate of person's catheter ablation is high.The postoperative periodic review for having carried out 1 year by a definite date of patient, Yu Shuhou September check hair
Existing atrial fibrillation recurrence.Therefore, the plasma TM AO assessment late recurrence after surgery risk height and expected one of the preoperative higher level of the patient
It causes.
Patient three
One, clinical data
Patient, male, 63 years old, because " palpitaition 1 year repeatedly " is admitted to hospital, diagnosis: auricular fibrillation (duration), row ring pulmonary vein isolation art,
Art mesostroma mapping, is shown in left room roof sheet low-voltage area and fragmented potential, and low-voltage area is isolated for parallel local host improvement.
Post-operative recovery sinus rhythm.It is postoperative to have no that atrial fibrillation is sent out again respectively at postoperative March, June, September, check in December.
Two, the measurement of plasma TM AO:
TMAO high performance liquid chromatography tandem mass spectrum kit measurement method: (1) the preoperative limosis vein blood 3ml of patient is acquired, is placed in
EDTA anti-coagulants vacuum tube, 4 DEG C of 3500r are centrifuged l0min, isolate blood plasma, set -80 DEG C of refrigerators and save.(2) kit is taken out
Configuration standard product: mother liquor: ultrapure water configures the standard TMAO stock solution of 1mg/ml, and -20 DEG C of refrigerator storages are spare.Removing protein liquid:
Ultrapure water configures the d9-TMAO internal standard solution of 0.7mg/ml, and with 500 times of dilution in acetonitrile, -20 DEG C of refrigerator storages are spare.(3)
Configuration standard curve: TMAO mother liquor is diluted step by step, be configured to various concentration standard solution (50,125,250,500,1250,
2500,5000ng/mL), removing protein liquid is added, whirlpool shakes 1min, and at 4 DEG C, 13000rpm is centrifuged 15min.Take supernatant into
Sample detection is drawn using sample introduction concentration as abscissa, and peak area is that ordinate draws standard curve.(4) patient's blood specimen is taken out,
It is placed in thaw at RT.20 μ L plasma samples are taken, 80 μ L removing protein liquid are added and are put into centrifuge tube, concussion 1min is sufficiently mixed
Even, 4 DEG C, 13000rpm is centrifuged 15min.Supernatant is taken to be transferred to sample injection bottle, machine testing in preparation.(5) it adjusts chromatographic condition: using
1502.1mm, 1.7 μm of hydrophilic chromatographic column；Mobile phase A: water (5mmol ammonium acetate)；Mobile phase B: acetonitrile.Using Gradient program
Elution, gradient condition: 0-5min, 50%A；5-10min, 50%A-80%, 10-15min, 80%A run total time 15min；Stream
Speed: 0.3mL/min；Sample volume: 3 μ L；Column temperature: 30 DEG C.(6) Mass Spectrometry Conditions: ion source: electric spray ion source (ESI) are adjusted, just
Ion mode；High pure nitrogen (purity 99.999%) is as dry gas, dry 325 DEG C of temperature degree, dry gas stream speed 10L/min；It sweeps
Retouch mode: multiple-reaction monitoring pattern (MRM), quota ion pair: TMAOm/z76 → 58, d9-TMAOm/z85 → 66；It is qualitative from
Son: TMAOm/z42, d9-TMAOm/z46；Collision energy: TMAO20eV, d9-TMAO25eV.(7) TMAO is detected: with d9-TMAO
It is quantified as Isotopic Internal Standard, by flowing A, B phase, obtains mass spectrogram, peak area is converted into chromatographic data through computer
Obtain patients blood plasma's sample TMAO concentration.As a result as shown in Figure 3 as a result, going out the preoperative sky of the patients with atrial fibrillation using this kits
Abdomen plasma TM AO content is 3.17 μm of ol/L.
Three, conclusion
The preoperative plasma TM AO content of patient is 3.17 μm of ol/L, similar to 2 grades of levels of plasma TM AO in embodiment 1, and prediction is suffered from
The postoperative late recurrent rate of person's catheter ablation is lower.The postoperative periodic review for having carried out 1 year by a definite date of patient, postoperative no atrial fibrillation are multiple
Hair.Therefore, the plasma TM AO assessment late recurrence after surgery risk of the preoperative reduced levels of the patient is low and expected consistent.
Patient four
One, clinical data
Patient, male 64 years old, because " palpitaition 1 year " is admitted to hospital, diagnose: auricular fibrillation (duration), row ring pulmonary vein isolation art, in art
Substrate-based mapping has no low-voltage area.Post-operative recovery sinus rhythm.Respectively at postoperative March, June, September, check in December, with postoperative 6
Moon discovery atrial fibrillation is sent out again.
Two, the measurement of plasma TM AO:
TMAO high performance liquid chromatography tandem mass spectrum kit measurement method: (1) the preoperative limosis vein blood 3ml of patient is acquired, is placed in
EDTA anti-coagulants vacuum tube, 4 DEG C of 3500r are centrifuged l0min, isolate blood plasma, set -80 DEG C of refrigerators and save.(2) kit is taken out
Configuration standard product: mother liquor: ultrapure water configures the standard TMAO stock solution of 1mg/ml, and -20 DEG C of refrigerator storages are spare.Removing protein liquid:
Ultrapure water configures the d9-TMAO internal standard solution of 0.7mg/ml, and with 500 times of dilution in acetonitrile, -20 DEG C of refrigerator storages are spare.(3)
Configuration standard curve: TMAO mother liquor is diluted step by step, be configured to various concentration standard solution (50,125,250,500,1250,
2500,5000ng/mL), removing protein liquid is added, whirlpool shakes 1min, and at 4 DEG C, 13000rpm is centrifuged 15min.Take supernatant into
Sample detection is drawn using sample introduction concentration as abscissa, and peak area is that ordinate draws standard curve.(4) patient's blood specimen is taken out,
It is placed in thaw at RT.20 μ L plasma samples are taken, 80 μ L removing protein liquid are added and are put into centrifuge tube, concussion 1min is sufficiently mixed
Even, 4 DEG C, 13000rpm is centrifuged 15min.Supernatant is taken to be transferred to sample injection bottle, machine testing in preparation.(5) it adjusts chromatographic condition: using
1502.1mm, 1.7 μm of hydrophilic chromatographic column；Mobile phase A: water (5mmol ammonium acetate)；Mobile phase B: acetonitrile.Using Gradient program
Elution, gradient condition: 0-5min, 50%A；5-10min, 50%A-80%, 10-15min, 80%A run total time 15min；Stream
Speed: 0.3mL/min；Sample volume: 3 μ L；Column temperature: 30 DEG C.(6) Mass Spectrometry Conditions: ion source: electric spray ion source (ESI) are adjusted, just
Ion mode；High pure nitrogen (purity 99.999%) is as dry gas, dry 325 DEG C of temperature degree, dry gas stream speed 10L/min；It sweeps
Retouch mode: multiple-reaction monitoring pattern (MRM), quota ion pair: TMAOm/z76 → 58, d9-TMAOm/z85 → 66；It is qualitative from
Son: TMAOm/z42, d9-TMAOm/z46；Collision energy: TMAO20eV, d9-TMAO25eV.(7) TMAO is detected: with d9-TMAO
It is quantified as Isotopic Internal Standard, by flowing A, B phase, obtains mass spectrogram, peak area is converted into chromatographic data through computer
Obtain patients blood plasma's sample TMAO concentration.As a result as shown in Figure 3 as a result, going out the preoperative sky of the patients with atrial fibrillation using this kits
Abdomen plasma TM AO content is 4.32 μm of ol/L.
Three, conclusion
The preoperative plasma TM AO content of patient is that 4.32 μm of ol/L(assume), it is similar to 3 grades of levels of plasma TM AO in embodiment 1,
Late recurrent rate after predicting patient catheter's ablation is high.The postoperative periodic review for having carried out 1 year by a definite date of patient, postoperative hair in June
Existing atrial fibrillation recurrence.Therefore, the preoperative high-caliber plasma TM AO of the patient assesses late recurrence after surgery risk height and is expected consistent.
Patient five
One, clinical data
Patient, female, 59 years old, because " ictal palpitaition 4 years " is admitted to hospital, diagnosis: auricular fibrillation (paroxysmal), row ring pulmonary vein isolation
Art, art mesostroma mapping, has no low-voltage area.Post-operative recovery sinus rhythm.It is checked respectively at postoperative March, June, September, December,
Do not find that atrial fibrillation is sent out again.
Two, the measurement of plasma TM AO:
TMAO high performance liquid chromatography tandem mass spectrum kit measurement method: (1) the preoperative limosis vein blood 3ml of patient is acquired, is placed in
EDTA anti-coagulants vacuum tube, 4 DEG C of 3500r are centrifuged l0min, isolate blood plasma, set -80 DEG C of refrigerators and save.(2) kit is taken out
Configuration standard product: mother liquor: ultrapure water configures the standard TMAO stock solution of 1mg/ml, and -20 DEG C of refrigerator storages are spare.Removing protein liquid:
Ultrapure water configures the d9-TMAO internal standard solution of 0.7mg/ml, and with 500 times of dilution in acetonitrile, -20 DEG C of refrigerator storages are spare.(3)
Configuration standard curve: TMAO mother liquor is diluted step by step, be configured to various concentration standard solution (50,125,250,500,1250,
2500,5000ng/mL), removing protein liquid is added, whirlpool shakes 1min, and at 4 DEG C, 13000rpm is centrifuged 15min.Take supernatant into
Sample detection is drawn using sample introduction concentration as abscissa, and peak area is that ordinate draws standard curve.(4) patient's blood specimen is taken out,
It is placed in thaw at RT.20 μ L plasma samples are taken, 80 μ L removing protein liquid are added and are put into centrifuge tube, concussion 1min is sufficiently mixed
Even, 4 DEG C, 13000rpm is centrifuged 15min.Supernatant is taken to be transferred to sample injection bottle, machine testing in preparation.(5) it adjusts chromatographic condition: using
1502.1mm, 1.7 μm of hydrophilic chromatographic column；Mobile phase A: water (5mmol ammonium acetate)；Mobile phase B: acetonitrile.Using Gradient program
Elution, gradient condition: 0-5min, 50%A；5-10min, 50%A-80%, 10-15min, 80%A run total time 15min；Stream
Speed: 0.3mL/min；Sample volume: 3 μ L；Column temperature: 30 DEG C.(6) Mass Spectrometry Conditions: ion source: electric spray ion source (ESI) are adjusted, just
Ion mode；High pure nitrogen (purity 99.999%) is as dry gas, dry 325 DEG C of temperature degree, dry gas stream speed 10L/min；It sweeps
Retouch mode: multiple-reaction monitoring pattern (MRM), quota ion pair: TMAOm/z76 → 58, d9-TMAOm/z85 → 66；It is qualitative from
Son: TMAOm/z42, d9-TMAOm/z46；Collision energy: TMAO20eV, d9-TMAO25eV.(7) TMAO is detected: with d9-TMAO
It is quantified as Isotopic Internal Standard, by flowing A, B phase, obtains mass spectrogram, peak area is converted into chromatographic data through computer
Obtain patients blood plasma's sample TMAO concentration.As a result as shown in Figure 3 as a result, going out the preoperative sky of the patients with atrial fibrillation using this kits
Abdomen plasma TM AO content is 1.89 μm of ol/L.
Three, conclusion
The preoperative plasma TM AO content of patient is 1.89 μm of ol/L, similar to 1 grade of level of plasma TM AO in embodiment 1, and prediction is suffered from
The postoperative late recurrent rate of person's catheter ablation is low.The postoperative periodic review for having carried out 1 year by a definite date of patient, postoperative June find atrial fibrillation
Recurrence.Therefore, the preoperative low-level plasma TM AO assessment late recurrence after surgery risk of the patient is low and expected consistent.
Patient six
One, clinical data
Patient, male, 47 years old, because " paroxysmal palpitaition 5 years " is admitted to hospital, diagnosis: auricular fibrillation (paroxysmal), row ring pulmonary vein isolation
Art, art mesostroma mapping, has no low-voltage area.Post-operative recovery sinus rhythm.It is checked respectively at postoperative March, June, September, December,
Postoperative December finds atrial fibrillation recurrence when checking.
Two, the measurement of plasma TM AO:
TMAO high performance liquid chromatography tandem mass spectrum kit measurement method: (1) the preoperative limosis vein blood 3ml of patient is acquired, is placed in
EDTA anti-coagulants vacuum tube, 4 DEG C of 3500r are centrifuged l0min, isolate blood plasma, set -80 DEG C of refrigerators and save.(2) kit is taken out
Configuration standard product: mother liquor: ultrapure water configures the standard TMAO stock solution of 1mg/ml, and -20 DEG C of refrigerator storages are spare.Removing protein liquid:
Ultrapure water configures the d9-TMAO internal standard solution of 0.7mg/ml, and with 500 times of dilution in acetonitrile, -20 DEG C of refrigerator storages are spare.(3)
Configuration standard curve: TMAO mother liquor is diluted step by step, be configured to various concentration standard solution (50,125,250,500,1250,
2500,5000ng/mL), removing protein liquid is added, whirlpool shakes 1min, and at 4 DEG C, 13000rpm is centrifuged 15min.Take supernatant into
Sample detection is drawn using sample introduction concentration as abscissa, and peak area is that ordinate draws standard curve.(4) patient's blood specimen is taken out,
It is placed in thaw at RT.20 μ L plasma samples are taken, 80 μ L removing protein liquid are added and are put into centrifuge tube, concussion 1min is sufficiently mixed
Even, 4 DEG C, 13000rpm is centrifuged 15min.Supernatant is taken to be transferred to sample injection bottle, machine testing in preparation.(5) it adjusts chromatographic condition: using
1502.1mm, 1.7 μm of hydrophilic chromatographic column；Mobile phase A: water (5mmol ammonium acetate)；Mobile phase B: acetonitrile.Using Gradient program
Elution, gradient condition: 0-5min, 50%A；5-10min, 50%A-80%, 10-15min, 80%A run total time 15min；Stream
Speed: 0.3mL/min；Sample volume: 3 μ L；Column temperature: 30 DEG C.(6) Mass Spectrometry Conditions: ion source: electric spray ion source (ESI) are adjusted, just
Ion mode；High pure nitrogen (purity 99.999%) is as dry gas, dry 325 DEG C of temperature degree, dry gas stream speed 10L/min；It sweeps
Retouch mode: multiple-reaction monitoring pattern (MRM), quota ion pair: TMAOm/z76 → 58, d9-TMAOm/z85 → 66；It is qualitative from
Son: TMAOm/z42, d9-TMAOm/z46；Collision energy: TMAO20eV, d9-TMAO25eV.(7) TMAO is detected: with d9-TMAO
It is quantified as Isotopic Internal Standard, by flowing A, B phase, obtains mass spectrogram, peak area is converted into chromatographic data through computer
Obtain patients blood plasma's sample TMAO concentration.As a result as shown in Figure 3 as a result, going out the preoperative sky of the patients with atrial fibrillation using this kits
Abdomen plasma TM AO content is 4.33 μm of ol/L.
Three, conclusion
The preoperative plasma TM AO content of patient is 4.33 μm of ol/L, similar to 3 grades of levels of plasma TM AO in embodiment 1, and prediction is suffered from
The postoperative late recurrent rate of person's catheter ablation is high.The postoperative periodic review for having carried out 1 year by a definite date of patient, postoperative December find atrial fibrillation
Recurrence.Therefore, the preoperative high-caliber plasma TM AO of the patient assesses late recurrence after surgery risk height and is expected consistent.
In conclusion kit proposed by the invention, for having actively for the postoperative recurrence of atrial fibrillation catheter ablation
Meaning.
Claims (4)
1. a kind of kit, it is characterised in that including following components: acetonitrile (chromatographically pure), ammonium acetate (chromatographically pure), TMAO standard
Product (purity >=99%), deuterated trimethylamine oxide (d9-TMAO) (purity >=98%).
2. kit according to claim 1, which is characterized in that the kit is used to detect the high performance liquid chromatography of TMAO
The kit of tandem mass spectrum.
3. kit according to claim 1, which is characterized in that the kit is for the postoperative recurrence of atrial fibrillation catheter ablation
Assessment.
4. kit according to claim 3, wherein the kit of the assessment for the postoperative recurrence of atrial fibrillation catheter ablation makes
With method are as follows:
A, the configuration of standard items: mother liquor: ultrapure water configures the standard TMAO stock solution of 1mg/ml, and -20 DEG C of refrigerator storages are spare, remove
Protein liquid: ultrapure water configures the d9-TMAO internal standard solution of 0.7mg/ml, and with 500 times of dilution in acetonitrile, -20 DEG C of refrigerator storages
It is spare；
B, the configuration of standard curve: detection before TMAO mother liquor is diluted step by step, be configured to various concentration standard solution (50,
125,250,500,1250,2500,5000ng/mL), the removing protein liquid in A is added, whirlpool shakes 1min, at 4 DEG C,
13000rpm is centrifuged 15min, takes supernatant sample detection, draws using sample introduction concentration as abscissa, and peak area is ordinate drafting
Standard curve；
C, the processing of plasma sample: taking 20 μ L plasma samples, and the removing protein liquid being added in the 80 above-mentioned A of μ L is put into centrifuge tube, shakes
It swings 1min to be sufficiently mixed uniformly, 4 DEG C, 13000rpm is centrifuged 15min, and supernatant is taken to be transferred to sample injection bottle, machine testing in preparation；
D, 1502.1mm, 1.7 μm of hydrophilic chromatographic column the adjusting of chromatographic condition: are used；Mobile phase A: water (5mmol ammonium acetate)；
Mobile phase B: acetonitrile is eluted using Gradient program, gradient condition: 0-5min, 50%A；5-10min, 50%A-80%, 10-15min,
80%A runs total time 15min；Flow velocity: 0.3mL/min；Sample volume: 3 μ L；Column temperature: 30 DEG C；
E, the adjusting of Mass Spectrometry Conditions: ion source: electric spray ion source (ESI), positive ion mode；High pure nitrogen (purity
99.999%) as dry gas, dry temperature degree 325C, dry gas stream speed 10L/min；Scanning mode: multiple-reaction monitoring pattern
(MRM), quota ion pair: TMAOm/z76 → 58, d9-TMAOm/z85 → 66；Qualitative ion: TMAOm/z42, d9-TMAOm/
z46；Collision energy: TMAO20eV, d9-TMAO25eV；
F, TMAO is detected: being quantified using d9-TMAO as Isotopic Internal Standard, by flowing A, B phase, obtains mass spectrogram, peak face
Product is converted into chromatographic data through computer and obtains test plasma sample TMAO concentration.
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| CN110208550A (en) * | 2019-07-03 | 2019-09-06 | 贵州省临床检验中心 | One kind marker relevant with risk of recurrence after Atrial fibrillation radiofrequency ablation is combined and its is applied |
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| CN107340341A (en) * | 2017-06-27 | 2017-11-10 | 长沙都正生物科技有限责任公司 | A kind of TMAO immue quantitative detection reagent box and method |
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| CN110208550A (en) * | 2019-07-03 | 2019-09-06 | 贵州省临床检验中心 | One kind marker relevant with risk of recurrence after Atrial fibrillation radiofrequency ablation is combined and its is applied |
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