CN109917042B - Kit - Google Patents

Abstract

The invention provides a kit suitable for evaluating the late recurrence risk after atrial fibrillation catheter ablation. Moreover, the kit is used for measuring the content of plasma TMAO, and the recurrence risk of patients with atrial fibrillation after catheter ablation can be effectively evaluated by measuring the TMAO in the plasma of the patients, so that doctors can conveniently evaluate the recurrence risk of patients after catheter ablation before operation, and further appoint a targeted treatment scheme, and timely and effective treatment is provided for the patients.

Description

Kit

Technical Field

The invention relates to the technical field of medical treatment, in particular to a kit.

Background

Atrial fibrillation is one of the most common clinical arrhythmias, and the incidence and prevalence rate thereof gradually increase with age. Stroke, heart failure, cardiac arrest and the like caused by atrial fibrillation can seriously increase the fatality rate and disability rate, and bring heavy medical burden. There are many treatments for atrial fibrillation, and catheter ablation is one of the important. However, the late recurrence rate after atrial fibrillation catheter ablation reaches 25% -40%, so that the assessment of the recurrence risk after atrial fibrillation catheter ablation and the assignment of a targeted treatment scheme before operation are of great significance.

The pathophysiological mechanisms of atrial fibrillation are not fully elucidated, and it is now generally believed that it is produced by initiation triggers and maintenance matrix interactions. Under the conditions of autonomic nerve stimulation, ischemia and the like, abnormal autonomic cells in the muscle cuff of the great cardiac vein (including pulmonary vein, vena cava and the like) can spontaneously generate rapid electrical activity, and the abnormal electrical activity is the initiation trigger factor of atrial fibrillation. The transmission of the initiation-triggered abnormal electrical activity to the atrial muscle triggers multiple reentry wavelets, resulting in the formation of atrial fibrillation. Atrial fibrillation is self-sustaining once triggered and relies on electrical and structural remodeling of the atria. The basis of electrical remodeling is that the gene expression of atrial myocytes can change under the conditions of atrial fibrillation, atrial stretch and the like, and further ion channels on the cell surface are changed. The change of ion channels on the surface of the cardiac muscle cells further causes the change of transmembrane ion current, such as the reduction of L-type calcium channel ion current, instantaneous outward potassium channel current and delayed rectification potassium current, and the increase of inward rectification potassium channel and ATP sensitive potassium channel current, and finally causes the shortening of effective refractory period, the increase of heterogeneity and the poor frequency adaptability of the atrial myocytes. Continued development of atrial fibrillation also leads to structural remodeling of the atria. The structural reconstruction caused by atrial fibrillation comprises gap connection reconstruction and cell reconstruction. Gap junction reconstitution is expressed as expression and distribution heterogeneity of connexin; cellular remodeling is atrial fibrosis induced by apoptosis of myocardial cells, myolysis, and proliferation of myocardial interstitial fibers. The change of the ultrastructure of atrial myocyte, myocardial fibrosis and redistribution of connexin can make activation slow and route tortuous, and finally promote the generation and maintenance of atrial fibrillation.

Researches show that atrial fibrillation can cause atrial electrical reconstruction and structural reconstruction, and then the atrial fibrillation-atrial fibrillation effect is shown, and the death rate and the disability rate can be seriously increased when the atrial fibrillation occurs, so that the significance of recovering the sinus rhythm of patients with atrial fibrillation is great. Among them, catheter ablation is one of the important means to restore sinus rhythm in patients with atrial fibrillation. More than 90% of trigger focuses detected in electrophysiological examination of patients with paroxysmal atrial fibrillation are located inside pulmonary veins, so pulmonary vein electrical isolation is the most basic and most common operation form for ablation of atrial fibrillation at present. However, the recurrence rate in the early postoperative period (within 3 months) reaches more than 50%, and fortunately, atrial fibrillation with early recurrence can disappear by itself within 3 months after operation, so that the ablation time after 3 months is used as a criterion for evaluating whether ablation is not late recurrence. The recurrence rate in the later period after operation (within 12 months) reaches 25-40%, and researches show that the probability of the first recurrence after 1 year of the ablation is very little, so that one year after the current period of the operation is the time most commonly used for evaluating the recurrence after the ablation of the atrial fibrillation catheter. Researches show that the postoperative late recurrence is related to the atrial remodeling degree, and the mapping in electrophysiological examination shows that the mechanism of the catheter postoperative late recurrence is the recanalization of pulmonary vein isolation and the triggering of a trigger focus outside the pulmonary vein, and previous researches show that TMAO can promote myocardial fibrosis by activating a TGF beta-P-SMAD 3 signal channel and shorten the effective refractory period of myocardial cells by activating an NF-kB signal channel, so that the TMAO has reason to believe that the TMAO can play a role in the atrial fibrillation catheter postoperative late recurrence.

With the continuous understanding of the pathogenesis of atrial fibrillation, the hazards related to atrial fibrillation and the recovery of the sinus rhythm of patients with atrial fibrillation are gradually paid attention to, and at present, no sensitive and specific biomarker for evaluating the risk of late recurrence after ablation of an atrial fibrillation catheter is still available, so that a method for evaluating the risk of late recurrence after ablation of the atrial fibrillation catheter, which is easy to detect and high in specificity, is urgently needed to be researched by experts and scholars in the medical field together for evaluating the risk of recurrence after the ablation of the atrial fibrillation catheter as early as possible before an operation, so that doctors can perform individualized treatment on patients (such as compound operative treatment on the basis of isolation of the pulmonary veins).

Disclosure of Invention

The invention aims to solve the defects in the prior art and provides a kit for evaluating recurrence after atrial fibrillation catheter ablation.

In order to achieve the purpose, the invention adopts the following technical scheme:

the kit comprises the following components: acetonitrile (chromatographically pure), ammonium acetate (chromatographically pure), TMAO standard substance (purity is more than or equal to 99 percent), deuterated trimethylamine oxide (d9-TMAO) (purity is more than or equal to 98 percent); the kit is used for detecting the high performance liquid chromatography tandem mass spectrum of the TMAO. And the kit is used for evaluating the recurrence after the atrial fibrillation catheter ablation.

The inventor of the invention finds a kit suitable for evaluating the late recurrence risk after atrial fibrillation catheter ablation through intensive research from the early stage of the disease onset of an atrial fibrillation patient to the post catheter ablation. Moreover, the kit is used for measuring the content of plasma TMAO, and the recurrence risk of patients with atrial fibrillation after catheter ablation can be effectively evaluated by measuring the TMAO in the plasma of the patients, so that doctors can conveniently evaluate the recurrence risk of patients after catheter ablation before operation, and further appoint a targeted treatment scheme, and timely and effective treatment is provided for the patients.

The kit for evaluating the late recurrence after the catheter ablation is used for evaluating the postoperative recurrence risk of a patient by measuring TMAO in plasma of the patient. TMAO is prepared by decomposing choline, lecithin and L-carnitine in food by intestinal microorganisms to generate trimethylamine, and then absorbing the trimethylamine into liver via hepatic portal vein system to oxidize flavin monooxygenase. After the TMAO enters blood, the cardiovascular system can generate various pathophysiological changes, such as the activation of a TGF beta-P-SMAD 3 signal channel to further promote myocardial fibrosis; the NF-kB signal channel is activated to increase the expression of inflammatory factors in the cardiac ganglion layer, so that the function and activity of ganglia are increased and the effective refractory period is shortened. The inventor finds that the expression of the plasma TMAO of the patient with atrial fibrillation is measured through high performance liquid chromatography-tandem mass spectrometry, and finds that the content of the plasma TMAO in the patient with atrial fibrillation relapsed after catheter ablation is obviously increased compared with the content of a non-late-stage relapsing group after catheter ablation, and the difference has statistical significance (the P is expected to be less than 0.05). The clinical research value of the plasma TMAO of the patient with the recurrence after the catheter ablation of the atrial fibrillation patient is higher, and the recurrence after the catheter ablation of the atrial fibrillation patient is closely related to the TMAO. The quantitative detection of the plasma TMAO of the atrial fibrillation patient can be used as an important basis for evaluating the late recurrence risk after catheter ablation and an evaluation index for formulating a reasonable individualized treatment scheme.

The TMAO evaluation kit, namely the TMAO high performance liquid chromatography tandem mass spectrometry kit, has the use method that:

and (3) configuring a standard product: mother liquor: preparing ultrapure water into a standard TMAO stock solution with the concentration of 1mg/ml, and storing the TMAO stock solution in a refrigerator at the temperature of-20 ℃ for later use. Protein removing liquid: the ultrapure water is prepared into d9-TMAO internal standard solution with the concentration of 0.7mg/ml, and is diluted by 500 times by acetonitrile and stored in a refrigerator at the temperature of-20 ℃ for later use.

Configuration of the standard curve: diluting TMAO mother liquor step by step before detection, preparing into standard solutions (50, 125, 250, 500, 1250, 2500, 5000 ng/mL) with different concentrations, adding the protein removing solution in A, vortexing for 1min, and centrifuging at 13000rpm for 15min at 4 deg.C. And drawing a standard curve by taking the sample injection concentration as a horizontal coordinate and the peak area as a vertical coordinate.

Processing of the sample: and (3) adding 80 mu L of the protein removing liquid in the A into 20 mu L of the plasma sample, placing into a centrifuge tube, shaking for 1min, fully and uniformly mixing, and centrifuging at 4 ℃ and 13000rpm for 15 min. Taking the supernatant, transferring the supernatant to a sample injection bottle, and preparing for detection on a computer.

Adjustment of chromatographic conditions: using an 1502.1mm, 1.7 μm hydrophilic column; mobile phase A: water (5mmol ammonium acetate); mobile phase B: and (3) acetonitrile. Elution was performed using a gradient program, gradient conditions: 0-5min, 50% A; 5-10min, 50% A-80%, 10-15min, 80% A, total operation time 15 min; flow rate: 0.3 mL/min; sample introduction amount: 3 mu L of the solution; column temperature: and (6) 30C.

Adjustment of mass spectrometry conditions: an ion source: electrospray ion source (ESI), positive ion mode; high-purity nitrogen (purity 99.999%) is used as a drying gas, the temperature of the drying gas is 325 ℃, and the flow rate of the drying gas is 10L/min; the scanning mode is as follows: multiple reaction monitoring mode (MRM), quantitative ion pairs: TMAAM/z 76 → 58, d 9-TMAAM/z 85 → 66; and (3) qualitative ion: TMAAM/z 42, d 9-TMAAM/z 46; collision energy: TMAO20eV, d9-TMAO25 eV.

TMAO detection: d9-TMAO is used as an isotope internal standard for quantification, formic acid and methanol are used as mobile A, B phases to obtain a mass spectrogram, and the peak area is converted into chromatographic data through a computer to obtain the TMAO concentration of the blood plasma sample to be detected.

Drawings

FIG. 1 is an EIC plot of TMAO in a blank solution;

FIG. 2 is an EIC diagram of d9-TMAO in a blank solution;

FIG. 3 is an EIC profile of TMAO in plasma;

FIG. 4 is an EIC plot of d9-TMAO in plasma;

FIG. 5 ROC curve of TMAO versus risk of late recurrence after atrial fibrillation catheter ablation.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.

Example 1: clinical study on advanced recurrence and plasma TMAO after atrial fibrillation catheter ablation

First, case selection

Selecting 60 patients with atrial fibrillation to be subjected to catheter ablation for the first time, wherein the operation form is pulmonary vein isolation operation, and respectively follow-up the recurrence conditions of the atrial fibrillation in 3 months, 6 months, 9 months and 12 months to be divided into a non-late recurrence group and a postoperative recurrence group; 30 patients with paroxysmal supraventricular tachycardia (30) were selected as the control group. [ exclusion criteria: the history of ablation of atrial fibrillation exists in the past; presence of left atrial thrombus; anticoagulation contraindication exists; history of myocardial infarction within six months; congestive heart failure; severe chronic organic disease; hyperthyroidism; a gestational period; psychiatric disorders

Second, measurement result of plasma TMAO

Before operation, 3ml of venous blood is extracted on an empty stomach in the early morning and placed in an EDTA anticoagulant vacuum tube, centrifuged at 3500rpm at 4 ℃ for l0min, plasma is separated out and stored in a refrigerator at 80 ℃ below zero. And (3) respectively and quantitatively determining the TMAO in the sample plasma by adopting a high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS).

The plasma TMAO values of 3 groups are shown in Table 1. The expression of TMAO in the postoperative recurrent group is obviously higher than that in the non-late recurrent group and the paroxysmal supraventricular tachycardia control group, and the difference has statistical significance (P < 0.05). There are literature reports that plasma TMAO levels can be divided into 4 grades, 1 grade: <2.43 umol/L; and 2, stage: 2.43 to 3.66 umol/L; and 3, level: 3.67 to 6.18 umol/L; 4, level: >6.18 umol/L. The postoperative recurrence group of this study was similar to the grade 3 level, the successful postoperative group was similar to the grade 1-2 level, and the control group was similar to the grade 1 level.

Table 1: comparison of 3 groups of plasma TMAO, TGF-. beta.and LA Tmsv-SD

Grouping		Control group	Non-advanced relapse group (n =41)	Postoperative recurrence group (n =19)
					Plasma TMAO (mu mol/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: left room asynchrony index. P <0.05 compared to non-late relapsing group.

In order to further evaluate the prediction value of the TMAO on the late recurrence after the catheter atrial fibrillation ablation, the late recurrence after the catheter atrial fibrillation ablation is taken as a diagnosis standard, the area of the TMAO under the ROC curve of the late recurrence after the catheter atrial fibrillation ablation is 0.875 by calculating the sensitivity and specificity indexes under different TMAO levels and drawing the ROC curve, and the high prediction value of the TMAO on the late recurrence after the catheter atrial fibrillation ablation is shown in figure 5.

Third, conclusion

The inventor of the invention discovers that the content of the plasma TMAO in a recurrent group after the catheter ablation of the atrial fibrillation is higher than that in a non-late recurrent group and a control group, which shows that the content of the plasma TMAO in the recurrent atrial fibrillation after the surgery has high clinical research value, and the recurrence condition after the catheter ablation of the atrial fibrillation is closely related to the plasma TMAO. In addition, the research also finds that plasma TGF-beta and left atrial asynchronous indexes of postoperative recurrence patients are obviously higher than those of non-late recurrence groups, and plasma TMAO is positively correlated with plasma TGF-beta and left atrial synchronous indexes, which indicates that TMAO is likely to promote myocardial fibrosis through a TGF beta-P-SMAD 3 signal channel, further leads to left atrial asynchronous activity and causes late recurrence after atrial fibrillation catheter ablation. The research discovers that the TMAO has medium and high prediction value on late recurrence after atrial fibrillation catheter ablation through ROC curve analysis.

Therefore, the plasma TMAO can be used as a sensitive evaluation index of late-stage relapse after atrial fibrillation catheter ablation. Detection of plasma TMAO may be used for pre-operative assessment of patients with atrial fibrillation to formulate personalized treatment regimens.

Example 2: specific examples of example 1

Patient one

First, clinical data

Patients, male, 53 years old, were admitted to the hospital for "palpitations more than half a year", diagnosed: atrial fibrillation (paroxysmal), circum-pulmonary vein isolation, intra-operative matrix mapping, no left atrial low voltage region was found. Sinus rhythm is restored after surgery. After the operation, the patients are reviewed for 3 months, 6 months, 9 months and 12 months respectively, and the atrial fibrillation does not recur.

Second, measurement of plasma TMAO:

the determination method of the TMAO high performance liquid chromatography tandem mass spectrometry kit comprises the following steps: (1) collecting 3ml of fasting venous blood before operation, placing in an EDTA anticoagulant vacuum tube, centrifuging at 3500r at 4 ℃ for l0min, separating out plasma, and storing in a refrigerator at-80 ℃. (2) Taking out the kit to prepare a standard substance: mother liquor: preparing ultrapure water into a standard TMAO stock solution with the concentration of 1mg/ml, and storing the TMAO stock solution in a refrigerator at the temperature of-20 ℃ for later use. Protein removing liquid: the ultrapure water is prepared into d9-TMAO internal standard solution with the concentration of 0.7mg/ml, and is diluted by 500 times by acetonitrile and stored in a refrigerator at the temperature of-20 ℃ for later use. (3) Configuring a standard curve: diluting TMAO mother liquor step by step, preparing into standard solutions (50, 125, 250, 500, 1250, 2500, 5000 ng/mL) with different concentrations, adding deproteinizing solution, vortex shaking for 1min, and centrifuging at 13000rpm for 15min at 4 deg.C. And taking the supernatant for sample injection detection, and drawing a standard curve by taking the sample injection concentration as a horizontal coordinate and taking the peak area as a vertical coordinate. (4) Taking out blood specimen of patient, and thawing at room temperature. Adding 80 μ L of protein removing solution into 20 μ L of plasma sample, placing into a centrifuge tube, shaking for 1min, mixing well, centrifuging at 4 deg.C and 13000rpm for 15 min. Taking the supernatant, transferring the supernatant to a sample injection bottle, and preparing for detection on a computer. (5) Adjusting chromatographic conditions: using an 1502.1mm, 1.7 μm hydrophilic column; mobile phase A: water (5mmol ammonium acetate); mobile phase B: and (3) acetonitrile. Elution was performed using a gradient program, gradient conditions: 0-5min, 50% A; 5-10min, 50% A-80%, 10-15min, 80% A, total operation time 15 min; flow rate: 0.3 mL/min; sample introduction amount: 3 mu L of the solution; column temperature: at 30 ℃. (6) Adjusting mass spectrum conditions: an ion source: electrospray ion source (ESI), positive ion mode; high-purity nitrogen (purity 99.999%) is used as a drying gas, the temperature of the drying gas is 325 ℃, and the flow rate of the drying gas is 10L/min; the scanning mode is as follows: multiple reaction monitoring mode (MRM), quantitative ion pairs: TMAAM/z 76 → 58, d 9-TMAAM/z 85 → 66; and (3) qualitative ion: TMAAM/z 42, d 9-TMAAM/z 46; collision energy: TMAO20eV, d9-TMAO25 eV. (7) Detecting TMAO: d9-TMAO is used as an isotope internal standard for quantification, a mass spectrogram is obtained by flowing A, B phases, and the peak area is converted into chromatographic data by a computer to obtain the TMAO concentration of the plasma sample of the patient. The result is shown in FIG. 3, and the fasting plasma TMAO content of the atrial fibrillation patient before operation is measured to be 2.2 mu mol/L by using the kit.

Third, conclusion

The patient pre-operative plasma TMAO levels were 2.2 μmol/L (assumed), similar to the plasma TMAO grade 1 levels in example 1, and predicted a low late stage recurrence rate after catheter ablation in the patient. The patients had a periodic review for 1 year after the operation without recurrence of atrial fibrillation. Thus, this patient had a low risk of late recurrence following pre-operative low levels of plasma TMAO assessment consistent with expectations.

Patient 2

First, clinical data

Patient, female, age 67, admitted for "repeated precordial discomfort for more than 1 month", diagnosed: atrial fibrillation (paroxysmal), pulmonary vein isolation in the row ring, and matrix mapping in the operation, see the left posterior atrial wall sheet-shaped low-voltage area, and the low-voltage area is isolated by parallel local matrix modification. Sinus rhythm is restored after surgery. And the reexamination is respectively carried out after 3 months, 6 months, 9 months and 12 months of operation, and the reexamination of atrial fibrillation is found after the reexamination is carried out after the 9 months of operation.

Second, measurement of plasma TMAO:

the determination method of the TMAO high performance liquid chromatography tandem mass spectrometry kit comprises the following steps: (1) collecting 3ml of fasting venous blood before operation, placing in an EDTA anticoagulant vacuum tube, centrifuging at 3500r at 4 ℃ for l0min, separating out plasma, and storing in a refrigerator at-80 ℃. (2) Taking out the kit to prepare a standard substance: mother liquor: preparing ultrapure water into a standard TMAO stock solution with the concentration of 1mg/ml, and storing the TMAO stock solution in a refrigerator at the temperature of-20 ℃ for later use. Protein removing liquid: the ultrapure water is prepared into d9-TMAO internal standard solution with the concentration of 0.7mg/ml, and is diluted by 500 times by acetonitrile and stored in a refrigerator at the temperature of-20 ℃ for later use. (3) Configuring a standard curve: diluting TMAO mother liquor step by step, preparing into standard solutions (50, 125, 250, 500, 1250, 2500, 5000 ng/mL) with different concentrations, adding deproteinizing solution, vortex shaking for 1min, and centrifuging at 13000rpm for 15min at 4 deg.C. And taking the supernatant for sample injection detection, and drawing a standard curve by taking the sample injection concentration as a horizontal coordinate and taking the peak area as a vertical coordinate. (4) Taking out blood specimen of patient, and thawing at room temperature. Adding 80 μ L of protein removing solution into 20 μ L of plasma sample, placing into a centrifuge tube, shaking for 1min, mixing well, centrifuging at 4 deg.C and 13000rpm for 15 min. Taking the supernatant, transferring the supernatant to a sample injection bottle, and preparing for detection on a computer. (5) Adjusting chromatographic conditions: using an 1502.1mm, 1.7 μm hydrophilic column; mobile phase A: water (5mmol ammonium acetate); mobile phase B: and (3) acetonitrile. Elution was performed using a gradient program, gradient conditions: 0-5min, 50% A; 5-10min, 50% A-80%, 10-15min, 80% A, total operation time 15 min; flow rate: 0.3 mL/min; sample introduction amount: 3 mu L of the solution; column temperature: at 30 ℃. (6) Adjusting mass spectrum conditions: an ion source: electrospray ion source (ESI), positive ion mode; high-purity nitrogen (purity 99.999%) is used as a drying gas, the temperature of the drying gas is 325 ℃, and the flow rate of the drying gas is 10L/min; the scanning mode is as follows: multiple reaction monitoring mode (MRM), quantitative ion pairs: TMAAM/z 76 → 58, d 9-TMAAM/z 85 → 66; and (3) qualitative ion: TMAAM/z 42, d 9-TMAAM/z 46; collision energy: TMAO20eV, d9-TMAO25 eV. (7) Detecting TMAO: d9-TMAO is used as an isotope internal standard for quantification, a mass spectrogram is obtained by flowing A, B phases, and the peak area is converted into chromatographic data by a computer to obtain the TMAO concentration of the plasma sample of the patient. The result is shown in FIG. 3, and the fasting plasma TMAO content of the atrial fibrillation patient before operation is measured to be 4.19 mu mol/L by using the kit.

Third, conclusion

The pre-operative plasma TMAO content of the patient was 4.19. mu. mol/L, similar to the plasma TMAO grade 3 level in example 1, and the patient was predicted to have a high late-stage recurrence rate after catheter ablation. After the operation, the patient is subjected to periodical reexamination for 1 year, and after 9 months of the operation, the relapse of atrial fibrillation is found. Thus, the higher level of plasma TMAO before surgery in this patient is consistent with the expectation that the risk of late recurrence after surgery is high.

Patient three

First, clinical data

Patient, male, age 63, was admitted to the hospital for "repeated palpitations for 1 year", and diagnosed: atrial fibrillation (persistence), circum-pulmonary vein isolation, intra-operative matrix mapping, see left parietal laminar low voltage regions and fractionated potentials, with parallel local matrix modification to isolate the low voltage regions. Sinus rhythm is restored after surgery. The reexamination is respectively carried out after 3 months, 6 months, 9 months and 12 months of the operation, and no atrial fibrillation occurs after the operation.

Second, measurement of plasma TMAO:

the determination method of the TMAO high performance liquid chromatography tandem mass spectrometry kit comprises the following steps: (1) collecting 3ml of fasting venous blood before operation, placing in an EDTA anticoagulant vacuum tube, centrifuging at 3500r at 4 ℃ for l0min, separating out plasma, and storing in a refrigerator at-80 ℃. (2) Taking out the kit to prepare a standard substance: mother liquor: preparing ultrapure water into a standard TMAO stock solution with the concentration of 1mg/ml, and storing the TMAO stock solution in a refrigerator at the temperature of-20 ℃ for later use. Protein removing liquid: the ultrapure water is prepared into d9-TMAO internal standard solution with the concentration of 0.7mg/ml, and is diluted by 500 times by acetonitrile and stored in a refrigerator at the temperature of-20 ℃ for later use. (3) Configuring a standard curve: diluting TMAO mother liquor step by step, preparing into standard solutions (50, 125, 250, 500, 1250, 2500, 5000 ng/mL) with different concentrations, adding deproteinizing solution, vortex shaking for 1min, and centrifuging at 13000rpm for 15min at 4 deg.C. And taking the supernatant for sample injection detection, and drawing a standard curve by taking the sample injection concentration as a horizontal coordinate and taking the peak area as a vertical coordinate. (4) Taking out blood specimen of patient, and thawing at room temperature. Adding 80 μ L of protein removing solution into 20 μ L of plasma sample, placing into a centrifuge tube, shaking for 1min, mixing well, centrifuging at 4 deg.C and 13000rpm for 15 min. Taking the supernatant, transferring the supernatant to a sample injection bottle, and preparing for detection on a computer. (5) Adjusting chromatographic conditions: using an 1502.1mm, 1.7 μm hydrophilic column; mobile phase A: water (5mmol ammonium acetate); mobile phase B: and (3) acetonitrile. Elution was performed using a gradient program, gradient conditions: 0-5min, 50% A; 5-10min, 50% A-80%, 10-15min, 80% A, total operation time 15 min; flow rate: 0.3 mL/min; sample introduction amount: 3 mu L of the solution; column temperature: at 30 ℃. (6) Adjusting mass spectrum conditions: an ion source: electrospray ion source (ESI), positive ion mode; high-purity nitrogen (purity 99.999%) is used as a drying gas, the temperature of the drying gas is 325 ℃, and the flow rate of the drying gas is 10L/min; the scanning mode is as follows: multiple reaction monitoring mode (MRM), quantitative ion pairs: TMAAM/z 76 → 58, d 9-TMAAM/z 85 → 66; and (3) qualitative ion: TMAAM/z 42, d 9-TMAAM/z 46; collision energy: TMAO20eV, d9-TMAO25 eV. (7) Detecting TMAO: d9-TMAO is used as an isotope internal standard for quantification, a mass spectrogram is obtained by flowing A, B phases, and the peak area is converted into chromatographic data by a computer to obtain the TMAO concentration of the plasma sample of the patient. The result is shown in FIG. 3, and the fasting plasma TMAO content of the atrial fibrillation patient before operation is measured to be 3.17 mu mol/L by using the kit.

Third, conclusion

The pre-operative plasma TMAO content of the patient was 3.17 μmol/L, similar to the plasma TMAO grade 2 level in example 1, and the late-stage recurrence rate after catheter ablation was predicted to be lower in the patient. The patients have periodic reexamination for 1 year after operation, and have no atrial fibrillation and relapse after operation. Thus, this patient had a low risk of late recurrence following the lower level of plasma TMAO assessment prior to surgery, consistent with expectations.

Patient four

First, clinical data

Patient, male, age 64, was admitted for "palpitations 1 year", diagnosed: atrial fibrillation (persistence), pulmonary vein isolation with annuli, intra-operative matrix mapping, no low voltage region seen. Sinus rhythm is restored after surgery. Reexamination is respectively carried out after 3 months, 6 months, 9 months and 12 months of operation, and atrial fibrillation is found after 6 months of operation.

Second, measurement of plasma TMAO:

the determination method of the TMAO high performance liquid chromatography tandem mass spectrometry kit comprises the following steps: (1) collecting 3ml of fasting venous blood before operation, placing in an EDTA anticoagulant vacuum tube, centrifuging at 3500r at 4 ℃ for l0min, separating out plasma, and storing in a refrigerator at-80 ℃. (2) Taking out the kit to prepare a standard substance: mother liquor: preparing ultrapure water into a standard TMAO stock solution with the concentration of 1mg/ml, and storing the TMAO stock solution in a refrigerator at the temperature of-20 ℃ for later use. Protein removing liquid: the ultrapure water is prepared into d9-TMAO internal standard solution with the concentration of 0.7mg/ml, and is diluted by 500 times by acetonitrile and stored in a refrigerator at the temperature of-20 ℃ for later use. (3) Configuring a standard curve: diluting TMAO mother liquor step by step, preparing into standard solutions (50, 125, 250, 500, 1250, 2500, 5000 ng/mL) with different concentrations, adding deproteinizing solution, vortex shaking for 1min, and centrifuging at 13000rpm for 15min at 4 deg.C. And taking the supernatant for sample injection detection, and drawing a standard curve by taking the sample injection concentration as a horizontal coordinate and taking the peak area as a vertical coordinate. (4) Taking out blood specimen of patient, and thawing at room temperature. Adding 80 μ L of protein removing solution into 20 μ L of plasma sample, placing into a centrifuge tube, shaking for 1min, mixing well, centrifuging at 4 deg.C and 13000rpm for 15 min. Taking the supernatant, transferring the supernatant to a sample injection bottle, and preparing for detection on a computer. (5) Adjusting chromatographic conditions: using an 1502.1mm, 1.7 μm hydrophilic column; mobile phase A: water (5mmol ammonium acetate); mobile phase B: and (3) acetonitrile. Elution was performed using a gradient program, gradient conditions: 0-5min, 50% A; 5-10min, 50% A-80%, 10-15min, 80% A, total operation time 15 min; flow rate: 0.3 mL/min; sample introduction amount: 3 mu L of the solution; column temperature: at 30 ℃. (6) Adjusting mass spectrum conditions: an ion source: electrospray ion source (ESI), positive ion mode; high-purity nitrogen (purity 99.999%) is used as a drying gas, the temperature of the drying gas is 325 ℃, and the flow rate of the drying gas is 10L/min; the scanning mode is as follows: multiple reaction monitoring mode (MRM), quantitative ion pairs: TMAAM/z 76 → 58, d 9-TMAAM/z 85 → 66; and (3) qualitative ion: TMAAM/z 42, d 9-TMAAM/z 46; collision energy: TMAO20eV, d9-TMAO25 eV. (7) Detecting TMAO: d9-TMAO is used as an isotope internal standard for quantification, a mass spectrogram is obtained by flowing A, B phases, and the peak area is converted into chromatographic data by a computer to obtain the TMAO concentration of the plasma sample of the patient. The result is shown in FIG. 3, and the fasting plasma TMAO content of the atrial fibrillation patient before operation is measured to be 4.32 mu mol/L by using the kit.

Third, conclusion

The patient pre-operative plasma TMAO levels were 4.32 μmol/L (assumed), similar to the plasma TMAO level 3 in example 1, and predicted a high rate of late-stage recurrence after catheter ablation in the patient. The patients are subjected to periodic review for 1 year after the operation, and the atrial fibrillation relapse is found after 6 months after the operation. Thus, this patient's preoperative high level of plasma TMAO assesses a high risk of late postoperative recurrence consistent with expectations.

Patient five

First, clinical data

Patient, female, 59 years old, admitted to hospital for "ictal palpitations for 4 years", diagnosed: atrial fibrillation (paroxysmal), isolation of the pulmonary veins in the annulus, and matrix mapping during the operation, and no low voltage region is seen. Sinus rhythm is restored after surgery. After the operation, the reexamination is respectively carried out for 3 months, 6 months, 9 months and 12 months, and no recurrent atrial fibrillation is found.

Second, measurement of plasma TMAO:

the determination method of the TMAO high performance liquid chromatography tandem mass spectrometry kit comprises the following steps: (1) collecting 3ml of fasting venous blood before operation, placing in an EDTA anticoagulant vacuum tube, centrifuging at 3500r at 4 ℃ for l0min, separating out plasma, and storing in a refrigerator at-80 ℃. (2) Taking out the kit to prepare a standard substance: mother liquor: preparing ultrapure water into a standard TMAO stock solution with the concentration of 1mg/ml, and storing the TMAO stock solution in a refrigerator at the temperature of-20 ℃ for later use. Protein removing liquid: the ultrapure water is prepared into d9-TMAO internal standard solution with the concentration of 0.7mg/ml, and is diluted by 500 times by acetonitrile and stored in a refrigerator at the temperature of-20 ℃ for later use. (3) Configuring a standard curve: diluting TMAO mother liquor step by step, preparing into standard solutions (50, 125, 250, 500, 1250, 2500, 5000 ng/mL) with different concentrations, adding deproteinizing solution, vortex shaking for 1min, and centrifuging at 13000rpm for 15min at 4 deg.C. And taking the supernatant for sample injection detection, and drawing a standard curve by taking the sample injection concentration as a horizontal coordinate and taking the peak area as a vertical coordinate. (4) Taking out blood specimen of patient, and thawing at room temperature. Adding 80 μ L of protein removing solution into 20 μ L of plasma sample, placing into a centrifuge tube, shaking for 1min, mixing well, centrifuging at 4 deg.C and 13000rpm for 15 min. Taking the supernatant, transferring the supernatant to a sample injection bottle, and preparing for detection on a computer. (5) Adjusting chromatographic conditions: using an 1502.1mm, 1.7 μm hydrophilic column; mobile phase A: water (5mmol ammonium acetate); mobile phase B: and (3) acetonitrile. Elution was performed using a gradient program, gradient conditions: 0-5min, 50% A; 5-10min, 50% A-80%, 10-15min, 80% A, total operation time 15 min; flow rate: 0.3 mL/min; sample introduction amount: 3 mu L of the solution; column temperature: at 30 ℃. (6) Adjusting mass spectrum conditions: an ion source: electrospray ion source (ESI), positive ion mode; high-purity nitrogen (purity 99.999%) is used as a drying gas, the temperature of the drying gas is 325 ℃, and the flow rate of the drying gas is 10L/min; the scanning mode is as follows: multiple reaction monitoring mode (MRM), quantitative ion pairs: TMAAM/z 76 → 58, d 9-TMAAM/z 85 → 66; and (3) qualitative ion: TMAAM/z 42, d 9-TMAAM/z 46; collision energy: TMAO20eV, d9-TMAO25 eV. (7) Detecting TMAO: d9-TMAO is used as an isotope internal standard for quantification, a mass spectrogram is obtained by flowing A, B phases, and the peak area is converted into chromatographic data by a computer to obtain the TMAO concentration of the plasma sample of the patient. The result is shown in FIG. 3, and the fasting plasma TMAO content of the atrial fibrillation patient before the operation is measured to be 1.89 mu mol/L by using the kit.

Third, conclusion

The pre-operative plasma TMAO content of the patient was 1.89. mu. mol/L, which was similar to the plasma TMAO grade 1 level in example 1, and predicted that the late-stage recurrence rate after catheter ablation in the patient was low. The patients are subjected to periodic review for 1 year after the operation, and the atrial fibrillation relapse is found after 6 months after the operation. Thus, this patient had a low risk of late recurrence following pre-operative low levels of plasma TMAO assessment consistent with expectations.

Six patients

First, clinical data

Patient, male, 47 years old, admitted to hospital for "paroxysmal palpitations for 5 years", diagnosed: atrial fibrillation (paroxysmal), isolation of the pulmonary veins in the annulus, and matrix mapping during the operation, and no low voltage region is seen. Sinus rhythm is restored after surgery. The relapse of atrial fibrillation is found in the reexamination of 3 months, 6 months, 9 months and 12 months after the operation and the reexamination of 12 months after the operation.

Second, measurement of plasma TMAO:

the determination method of the TMAO high performance liquid chromatography tandem mass spectrometry kit comprises the following steps: (1) collecting 3ml of fasting venous blood before operation, placing in an EDTA anticoagulant vacuum tube, centrifuging at 3500r at 4 ℃ for l0min, separating out plasma, and storing in a refrigerator at-80 ℃. (2) Taking out the kit to prepare a standard substance: mother liquor: preparing ultrapure water into a standard TMAO stock solution with the concentration of 1mg/ml, and storing the TMAO stock solution in a refrigerator at the temperature of-20 ℃ for later use. Protein removing liquid: the ultrapure water is prepared into d9-TMAO internal standard solution with the concentration of 0.7mg/ml, and is diluted by 500 times by acetonitrile and stored in a refrigerator at the temperature of-20 ℃ for later use. (3) Configuring a standard curve: diluting TMAO mother liquor step by step, preparing into standard solutions (50, 125, 250, 500, 1250, 2500, 5000 ng/mL) with different concentrations, adding deproteinizing solution, vortex shaking for 1min, and centrifuging at 13000rpm for 15min at 4 deg.C. And taking the supernatant for sample injection detection, and drawing a standard curve by taking the sample injection concentration as a horizontal coordinate and taking the peak area as a vertical coordinate. (4) Taking out blood specimen of patient, and thawing at room temperature. Adding 80 μ L of protein removing solution into 20 μ L of plasma sample, placing into a centrifuge tube, shaking for 1min, mixing well, centrifuging at 4 deg.C and 13000rpm for 15 min. Taking the supernatant, transferring the supernatant to a sample injection bottle, and preparing for detection on a computer. (5) Adjusting chromatographic conditions: using an 1502.1mm, 1.7 μm hydrophilic column; mobile phase A: water (5mmol ammonium acetate); mobile phase B: and (3) acetonitrile. Elution was performed using a gradient program, gradient conditions: 0-5min, 50% A; 5-10min, 50% A-80%, 10-15min, 80% A, total operation time 15 min; flow rate: 0.3 mL/min; sample introduction amount: 3 mu L of the solution; column temperature: at 30 ℃. (6) Adjusting mass spectrum conditions: an ion source: electrospray ion source (ESI), positive ion mode; high-purity nitrogen (purity 99.999%) is used as a drying gas, the temperature of the drying gas is 325 ℃, and the flow rate of the drying gas is 10L/min; the scanning mode is as follows: multiple reaction monitoring mode (MRM), quantitative ion pairs: TMAAM/z 76 → 58, d 9-TMAAM/z 85 → 66; and (3) qualitative ion: TMAAM/z 42, d 9-TMAAM/z 46; collision energy: TMAO20eV, d9-TMAO25 eV. (7) Detecting TMAO: d9-TMAO is used as an isotope internal standard for quantification, a mass spectrogram is obtained by flowing A, B phases, and the peak area is converted into chromatographic data by a computer to obtain the TMAO concentration of the plasma sample of the patient. The result is shown in FIG. 3, and the fasting plasma TMAO content of the atrial fibrillation patient before operation is measured to be 4.33 mu mol/L by using the kit.

Third, conclusion

The pre-operative plasma TMAO content of the patient was 4.33. mu. mol/L, similar to the plasma TMAO grade 3 level in example 1, and the patient was predicted to have a high late-stage recurrence rate after catheter ablation. After the operation, the patient is subjected to periodical reexamination for 1 year, and the atrial fibrillation relapse is found after 12 months of the operation. Thus, this patient's preoperative high level of plasma TMAO assesses a high risk of late postoperative recurrence consistent with expectations.

In conclusion, the kit provided by the invention has positive significance for recurrence after atrial fibrillation catheter ablation.

Claims (3)

1. The application of TMAO in preparing a kit for evaluating recurrence after ablation of atrial fibrillation catheters is characterized by comprising the following components: acetonitrile, ammonium acetate, TMAO standard, d 9-TMAO; the acetonitrile and ammonium acetate are chromatographically pure; the purity of the TMAO standard substance is more than or equal to 99 percent; the purity of the d9-TMAO is more than or equal to 98 percent.
2. 2. The use of claim 1, wherein the kit is for high performance liquid chromatography tandem mass spectrometry (HPLC-MS) for detecting TMAO.
3. 3. The use according to claim 1, characterized in that the kit is used in a method comprising:

   A. and (3) configuring a standard product: mother liquor: preparing 1mg/ml standard TMAO stock solution by ultrapure water, storing for later use in a refrigerator at the temperature of-20 ℃, removing protein solution: preparing ultrapure water into d9-TMAO internal standard solution of 0.7mg/ml, diluting with acetonitrile by 500 times, and storing in a refrigerator at-20 ℃ for later use;

   B. configuration of the standard curve: diluting the TMAO mother solution step by step before detection to prepare standard solutions with different concentrations, wherein the concentrations are as follows: 50. 125, 250, 500, 1250, 2500 and 5000ng/mL, adding the deproteinizing solution in the A, carrying out vortex oscillation for 1min, centrifuging for 15min at 4 ℃ and 13000rpm, taking supernate for sample injection detection, and drawing a standard curve by taking the sample injection concentration as a horizontal coordinate and taking a peak area as a vertical coordinate;

   C. treatment of plasma samples: adding 80 μ L of the protein removing liquid in A into 20 μ L of the plasma sample, placing into a centrifuge tube, shaking for 1min, mixing thoroughly, centrifuging at 4 deg.C and 13000rpm for 15min, taking the supernatant, transferring to a sample bottle, and preparing for detection on a computer;

   D. adjustment of chromatographic conditions: a 150 x 2.1mm, 1.7 μm hydrophilic column was used; mobile phase A: 5mmol/L ammonium acetate; mobile phase B: acetonitrile, elution using a gradient program, gradient conditions: 0-5min, 50% A; 5-10min, 50% A-80%, 10-15min, 80% A, total operation time 15 min; flow rate: 0.3 mL/min; sample introduction amount: 3 mu L of the solution; column temperature: 30 ℃;

   E. adjustment of mass spectrometry conditions: an ion source: electrospray ion source, positive ion mode; high-purity nitrogen is used as a drying gas, and the purity of the nitrogen is 99.999 percent; the temperature of the drying gas is 325 ℃, and the flow rate of the drying gas is 10L/min; the scanning mode is as follows: multiple reaction monitoring mode, quantitative ion pairs: TMAAM/z 76 → 58, d 9-TMAAM/z 85 → 66; and (3) qualitative ion: TMAAM/z 42, d 9-TMAAM/z 46; collision energy: TMAO20eV, d9-TMAO25 eV;

   F. TMAO detection: d9-TMAO is used as an isotope internal standard for quantification, a mass spectrogram is obtained by flowing A, B phases, and the peak area is converted into chromatographic data by a computer to obtain the TMAO concentration of the blood plasma sample to be detected.

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