CN117030874A - Application of fecal short-chain fatty acid as primary tremor diagnosis marker - Google Patents
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Abstract
The application provides application of fecal short chain fatty acid as a primary tremor diagnosis marker. The fecal short-chain fatty acid distinguishing the primary tremor from healthy people is any one or a combination of more of propionic acid, butyric acid and isobutyric acid; further, the fecal short-chain fatty acid distinguishing essential tremor from parkinsonism is any one or a combination of isobutyric acid and isovaleric acid. The application firstly provides that the difference of the fecal short-chain fatty acid of the primary tremor patient, the Parkinson disease patient and the healthy person is found by detecting the fecal short-chain fatty acid, and the differential fecal short-chain fatty acid is screened out to be used as a diagnosis marker of the primary tremor, so that the differential fecal short-chain fatty acid can be used for differential diagnosis of the primary tremor and the Parkinson disease, and has important significance for early diagnosis, early intervention, deep understanding of pathological mechanisms of the primary tremor and the Parkinson disease and the like. Meanwhile, the application takes the feces as the sample, and compared with cerebrospinal fluid and other samples, the application has the advantages of easy acquisition, convenient collection, large quantity, low price compared with neural images and short detection period.
Description
Technical Field
The application belongs to the technical field of biological diagnosis, and particularly relates to application of fecal short-chain fatty acid as a primary tremor diagnosis marker for differential diagnosis of parkinsonism.
Background
Essential tremor (EssentialTremor, ET) is a progressive, chronic, neurodegenerative disease that manifests itself primarily as actionable tremors of the upper extremities, which can involve other parts of the body, such as the head, vocal cords and lower extremities. The clinical features of ET include not only motor symptoms but also some non-motor features such as gastrointestinal dysfunction, mild cognitive impairment, mild gait dyskinesia, anxiety and depression. The pathological features of primary tremor are that there is a lewy body in blue spot nucleus or a torpedo-like change in cerebellar purkinje cells, and the etiology and pathogenesis of the primary tremor involve factors in both genetic and environmental aspects.
Parkinson's Disease (PD) and primary tremor are common tremor types in clinic, and since Parkinson's disease is a degenerative disease of the nervous system, the two diseases overlap in clinical symptoms, clinical manifestations are often very similar, and it is difficult to distinguish the disease by clinical symptoms alone, especially in early stages of the disease. The primary tremor and the parkinsonism are two diseases with completely different treatment and prognosis, and misdiagnosis not only delays diagnosis of the disease, but also brings unnecessary burden to the spirit and economy of the patient, so that early correct diagnosis is important. In summary, on the one hand, there is a need to find sensitive and reliable biological markers, and on the other hand, there is a need for an easy-to-operate and economical auxiliary examination means to provide assistance for diagnosis and differential diagnosis of essential tremors.
The existing biological marker research on primary tremor diagnosis and differential diagnosis is mainly focused on clinical symptoms, neuroimaging, neuroelectrophysiology and biochemistry: (1) clinical symptoms: parkinson's disease is mainly characterized by resting tremor, myotonia, slow motion and abnormal posture gait. However, in early stage and mild patients, there is a clinical overlap with the primary tremor patients, and the diagnosis is difficult, subjective, standardized and discriminated from the clinical manifestations. (2) neuroimaging: the known imaging methods for primary tremor diagnosis and parkinsonism differential diagnosis at present comprise skull ultrasound, CT, MRI and the like, but single imaging symptoms do not have independent high sensitivity and specificity, multiple imaging manifestations are needed to be integrated to improve the accuracy of diagnosis, and the imaging method is difficult to popularize due to high price and low practicality. Among them, studies have been made on identification using functional imaging of dopamine transporter, but it is difficult to apply them widely in clinic because of high requirements for examination equipment, high cost, or the need to use radiolabeled tracers. (3) neuroelectrophysiology: electromyographic tremor analysis can be used for identifying tremor types by carrying out more accurate quantitative analysis on tremor through specific tremor characteristics (including tremor frequency, amplitude, frequency spectrum harmonic wave, muscle contraction mode and the like), but single nerve electrophysiological examination does not have independent high sensitivity and specificity, other methods are needed to be integrated to improve diagnosis accuracy, nerve electrophysiological operation has stronger specialty, technician operation heterogeneity of different backgrounds is larger, and therefore popularization and application are affected to a certain extent. (4) biochemistry: the research shows that alpha-synuclein, DJ-1, dopamine metabolite, oxidative stress related factors, inflammatory factors and the like can be used for distinguishing primary tremors from parkinsonism, but the heterogeneity of results among the researches is large, particularly the heterogeneity of peripheral body fluid biomarkers is large, the sensitivity and specificity of a single body fluid marker are low, and the specificity and sensitivity of diagnosis and differential diagnosis can be improved by combining two or more markers. Gene detection can also be used for differential diagnosis, but has high cost and is difficult to popularize and apply in clinic. Therefore, it is required to find a biological marker for primary tremor diagnosis and differential diagnosis which has high sensitivity and specificity and can be widely applied in clinic.
Intestinal flora is the most important functional component in the intestinal tract and has important effects on human health and diseases. In recent years, the role played by intestinal flora in the intestinal-brain axis has been gradually recognized and has become a research hotspot, and research has shown that dysfunction of intestinal microorganisms-the intestinal-brain axis may lead to neurodegenerative diseases, and more evidence has suggested that there is a potential bidirectional relationship between intestinal microorganisms and neurodegenerative diseases. Notably, fecal microbiota transplantation improved both tremor and irritable bowel syndrome in primary tremor patients, possibly indicating a close relationship between intestinal microbiota and primary tremor. Furthermore, we found specific changes in intestinal microbiota in primary tremor patients, which strongly demonstrated that intestinal dysbacteriosis plays an important role in the onset of primary tremor. In addition to essential tremors, numerous studies have also shown that there are also disorders of the intestinal flora and their metabolites in parkinson's disease patients in different populations. However, primary tremor and parkinson's disease have different changes in intestinal flora, suggesting that the metabolic pathogenic pattern of the intestinal flora of the two diseases may be different.
Short Chain Fatty Acids (SCFAs), produced mainly by fermentation of dietary fibers by colonic bacteria, play an important role in regulating intestinal homeostasis and immune function and maintaining tight junctions of intestinal walls, and more studies have found that SCFAs can be absorbed by the intestinal tract, pass through the portal venous system to the liver to enter the blood circulation, and the SCFAs-producing bacteria in the intestinal tract strengthen the blood brain barrier function by promoting the expression of tight junctions of the blood brain barrier, which can affect host metabolism and immunity. At present, flora change in the feces of patients with primary tremor is reported, but change of SCFAs in the feces of patients with primary tremor is not reported. Given the important role of the gut microbiota-gut-brain axis in neurodegenerative diseases, and the potential role of the gut microbiota metabolite SCFAs, SCFAs are potential to be early diagnostic and differential diagnostic biomarkers for essential tremor.
Disclosure of Invention
The application is based on the above research and improves the diagnosis of the following defects of the prior art: (1) In early-stage and mild patients, the primary tremor and the parkinsonism patients have clinical symptoms overlapped, so that the diagnosis difficulty is high, the subjectivity is high, the standardization is difficult, and the distinction is difficult by clinical manifestation alone; (2) Single imaging symptoms do not have single high sensitivity and specificity, and imaging methods are difficult to widely apply in clinic due to high price, high requirement of inspection equipment, or the need of using radiolabeled tracers; (3) The single nerve electrophysiological examination has no single high sensitivity and specificity, and the operation has stronger specialty, and the popularization and the application are affected to a certain extent; (4) The heterogeneity of biochemical results is greater and single body fluid markers are less sensitive and specific.
The first object of the application is to provide the application of fecal short-chain fatty acid as a primary tremor diagnosis and differential diagnosis marker, and provide an auxiliary detection means which is simple and economical to operate for the diagnosis of primary tremor and the differential diagnosis of the primary tremor and parkinsonism.
The second object of the present application is to provide the use of the primary tremor diagnosis marker in the preparation of a primary tremor product for differential diagnosis with healthy persons or parkinson's disease.
The third object of the application is to provide a diagnostic kit for primary tremor and a diagnostic system for primary tremor based on the judgment of gas chromatography-mass spectrometry detection data for establishing the primary tremor diagnosis and the model for differential diagnosis of primary tremor and parkinsonism.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows: stool samples from primary tremor patients, parkinson's disease patients, and Healthy Controls (HC) were collected in several portions; extracting short chain fatty acids from the fecal sample based on gas chromatography-mass spectrometry (Gas chromatography-Massspectrometry, GC-MS), respectively; comparing the fecal short-chain fatty acid concentrations of the different samples of the step, and screening differential fecal short-chain fatty acids of primary tremor patients and HC and Parkinson disease patients; and (3) analyzing the working characteristic curve of the subject on the differential fecal short-chain fatty acid, and determining that fecal short-chain fatty acid propionic acid, butyric acid and isobutyric acid are used as primary tremor diagnosis markers, wherein isovaleric acid and isobutyric acid can be used for differential diagnosis of parkinsonism.
In a first aspect of the application, there is provided the use of a reagent for detecting fecal short chain fatty acids in the manufacture of a primary tremor diagnostic reagent or kit, wherein the fecal short chain fatty acids are any one or a combination of more of propionic acid, butyric acid, isobutyric acid, isovaleric acid.
The fecal short-chain fatty acid distinguishing the primary tremor from healthy people is any one or a combination of more of propionic acid, butyric acid and isobutyric acid; further, the fecal short-chain fatty acid distinguishing essential tremor from parkinsonism is any one or a combination of isobutyric acid and isovaleric acid.
Based on the analysis of the working characteristic curves of the differential short-chain fatty acids, the short-chain fatty acid markers can be used as diagnosis markers independently or can be combined for diagnosis.
When differential diagnosis is performed with healthy people, the intercept point concentration with propionic acid as a diagnosis marker is 675.727 mug/g, the intercept point concentration with butyric acid as a diagnosis marker is 1502.558 mug/g, and the intercept point concentration with isobutyric acid as a diagnosis marker is 94.236 mug/g:
the model for identifying essential tremors in healthy people is as follows:
Y1＝2.835-0.001*A-0.001*B-0.013*C，Prob1＝e Y1 /(1+e Y1 )；
wherein A, B, C is the concentration value of propionic acid, butyric acid and isobutyric acid detected by gas chromatography-mass spectrometry in the fecal sample; prob1 represents the probability of judging essential tremor.
Further, when differential diagnosis is performed on the parkinsonism, the differential markers of fecal short-chain fatty acid are isovaleric acid and isobutyric acid, the concentration of the intercept point of the isovaleric acid serving as a diagnostic marker is 79.179 mug/g, and the concentration of the intercept point of the isobutyric acid serving as a diagnostic marker is 107.871 mug/g;
the model for differential diagnosis of essential tremor and parkinson's disease is as follows:
Y2＝-2.100-0.042*D+0.067*E，Prob2＝e Y2 /(1+e Y2 )；
wherein D, E is the concentration value of isovaleric acid and isobutyric acid obtained by gas chromatography-mass spectrometry detection in the fecal sample, and Prob2 represents the probability of judging primary tremor.
Further, in a second aspect of the application, there is provided the use of a reagent for detecting fecal short chain fatty acid, either isobutyric acid, isovaleric acid or a combination of both, in the preparation of a reagent or kit for identifying essential tremor and parkinson's disease. The cut-off concentration of the marker and the occurrence probability calculation model of the primary tremor are the same as above.
Preferably, in both aspects, the step of detecting short chain fatty acids in the fecal sample is as follows:
(1) Taking a certain amount of fecal sample in a centrifuge tube, sequentially adding 15% phosphoric acid, 125 mug/ml internal standard isohexide solution and diethyl ether for homogenating, and centrifuging at 12000rpm at 4 ℃ for 10min to obtain supernatant;
(2) Short-chain fatty acid detection is carried out by adopting a ThermoTrace1310 gas chromatography-mass spectrometer, and target short-chain fatty acid and internal standard isocaproic acid are extracted, wherein the gas chromatography-mass spectrometer has the following conditions:
chromatographic column: an AgilentHP-INNOWAX capillary column; heating program: the initial temperature is kept at 90 ℃ for 1 minute, and is raised to 120 ℃ at 10 ℃/min, then raised to 150 ℃ at 5 ℃/min, and finally raised to 250 ℃ at 25 ℃/min for 2 minutes; carrier gas: helium, flow rate 1.0mL/min; the temperature of the sample inlet is 250 ℃, the sample is split and injected at a ratio of 10:1, and the sample injection volume is 1 mu L;
mass spectrometry conditions: electron ionization source with electron energy of 70eV; the temperature of the gas chromatography mass spectrometry interface is 250 ℃ and the temperature of the ion source is 300 ℃; scanning mode: a single ion scanning mode;
(3) Raw data generated by GC-MS were peak integrated, corrected and quantified for each fecal short chain fatty acid using MassHunter software, and the peak area was used to calculate metabolite concentrations.
Based on the detection method of short chain fatty acid in the fecal sample, a third aspect of the application provides a primary tremor diagnosis kit comprising a fecal sample treatment reagent and an internal standard reagent. The fecal sample treatment reagent comprises 15% phosphoric acid and diethyl ether; the internal standard reagent is an isohexide solution of 125 mug/ml.
In a fourth aspect of the present application, there is provided a primary tremor diagnosis system comprising:
the input display module is used for inputting basic information of a patient and detection concentration of propionic acid, butyric acid, isobutyric acid and isovaleric acid in a fecal sample, and displaying a diagnosis result;
the calculation module calculates the probability of suffering from the essential tremor based on a preset model formula 1 and a preset model formula 2:
model equation 1: y1=2.835-0.001 a-0.001 b-0.013 c, prob1=e Y1 /(1+e Y1 )；
Wherein A, B, C is the concentration value of propionic acid, butyric acid and isobutyric acid detected by gas chromatography-mass spectrometry in the fecal sample; prob1 represents the probability of judging essential tremor;
model equation 2: y2= -2.100-0.042×d+0.067×e, prob2=e Y2 /(1+e Y2 )；
Wherein D, E is the concentration value of isovaleric acid and isobutyric acid obtained by gas chromatography-mass spectrometry detection in the fecal sample, and Prob2 represents the probability of judging primary tremor;
the judging module judges whether the primary tremor is suffered from based on preset rules and transmits the result to the input display module, and the rules are as follows: when the propionic acid concentration is not higher than 675.727 mug/g, the butyric acid concentration is not higher than 1502.558 mug/g, the isobutyric acid concentration is not higher than 94.236 mug/g or Prob1 is greater than 0.5, the essential tremor is judged; further, when the concentration of isovaleric acid is not higher than 79.179. Mu.g/g, the concentration of isobutyric acid is not higher than 107.871. Mu.g/g or Prob2 is more than 0.5, it is judged that essential tremor is not Parkinson's disease.
The storage module is used for storing various information in the running process of the primary tremor diagnosis system;
the control module controls the input display module, the calculation module and the judgment module to operate normally.
In a fifth aspect of the application, there is provided an in-built electronic device in a gas chromatograph-mass spectrometer comprising a data storage, a data processor and a computer program stored on the memory and executable on the processor of a primary tremor diagnostic system. The data memory stores detection conditions of gas chromatography-mass spectrometry, raw data generated by GC-MS and concentration values after processing and calculation.
And (3) placing a diagnosis program of the primary tremor diagnosis system in a control system of a gas chromatography-mass spectrometry device, directly analyzing the concentration data of the target short-chain fatty acid after the fecal sample is detected, and displaying a diagnosis result.
In a sixth aspect of the present application, there is provided a non-transitory computer readable storage medium having stored thereon a computer program for execution by a processor of implementing steps of a primary tremor diagnosis system. The computer program can be implanted into different control systems according to requirements, and the concentration data of the short chain fatty acid of an experimental target is processed and analyzed.
The application has the beneficial effects that:
(1) The application firstly provides that the differences between the fecal short-chain fatty acid and the fecal short-chain fatty acid of the primary tremor patient and the HC patient are discovered through detecting the fecal short-chain fatty acid, and the differential fecal short-chain fatty acid is screened out to be used as a diagnosis marker of the primary tremor, so that the differential fecal short-chain fatty acid can be used for differential diagnosis of the primary tremor and the Parkinson disease, and has important significance for early diagnosis, early intervention, deep understanding of pathological mechanisms of the primary tremor and the Parkinson disease and the like.
(2) The application takes the feces as the sample, and compared with cerebrospinal fluid and other samples, the application has the advantages of easy acquisition, convenient collection, large quantity, low price compared with neural images and short detection period. The differential diagnosis can be carried out by detecting the fecal markers, is suitable for large-scale clinical diagnosis of primary tremors in the population in China, assists in differential diagnosis of primary tremors and parkinsons, and provides a new thought and direction for primary tremors diagnosis.
(3) The application has the advantages of simple operation, low cost, short detection time, high sensitivity and strong specificity.
Drawings
FIG. 1 is a flow chart of a screening method for assisting in differential feces short-chain fatty acid differential diagnosis of primary tremors, parkinson's disease and healthy people in the embodiment of the application;
figure 2 comparison of the differences in expression of the differential short chain fatty acids screened in essential tremor, parkinson's disease, healthy people: a. c, g, propionic acid, butyric acid and isobutyric acid differ in primary tremor patients and healthy people; d. g: iso-valeric acid and iso-butyric acid differ in primary tremor patients and parkinsonism patients;
fig. 3 stool short chain fatty acids distinguish primary tremors from healthy humans, primary tremors from parkinson's disease subject work profiles (ROC profile for receiver operatingchallenge): a, propionic acid, butyric acid and isobutyric acid distinguish 37 primary tremor patients from 35 healthy human results; b, isovaleric acid and isobutyric acid distinguish 37 cases of primary tremors from 37 cases of parkinsonism;
FIG. 4 is a schematic diagram of the primary tremor diagnosis system;
fig. 5 is a diagnostic flow chart of the essential tremor diagnostic system.
Detailed Description
The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application.
Screening for differential fecal short chain fatty acids
The fecal short chain fatty acid is determined by clinical experiment screening to be used as a diagnostic marker of primary tremor, can be used for differential diagnosis of parkinsonism, is approved by the ethical committee of the medical auxiliary ruijin hospital of Shanghai university in clinical experiments, and all subjects voluntarily participate in the clinical experiments are orally informed of the purpose of the experiments and sign informed consent.
1. The experimental object: primary tremor patients, parkinson's disease patients and healthy persons were collected from the clinic. The primary tremor patient group-entering criteria include clinically diagnosed primary tremor patients, and the diagnostic criteria are referred to the international movement disorder association primary tremor diagnostic expert consensus. The parkinsonian patient group-entering standard comprises clinically diagnosed primary parkinsonian patients, and the diagnosis standard refers to the international dyskinesia association parkinsonism diagnosis standard; other grouping criteria include: (1) age 25-85, (2) all parkinson's disease subjects were not dosed with antiparkinsonian drugs prior to collecting the stool samples, (3) the primary tremor group was dosed with beta-blockers alone or without related drug intake prior to collecting the stool samples. Healthy controls matched for age, sex, and body mass index (BodyMassIndex, BMI) were also selected.
The exclusion criteria were as follows: (1) vegetarian diet, (2) malnutrition, (3) chronic gastrointestinal disease (including inflammatory bowel disease, gastric ulcer or duodenal ulcer), (4) severe chronic disease (including malignancy, heart failure, renal insufficiency, blood system disease, hypertension, diabetes, etc), (5) major gastrointestinal surgical history, (6) continuous or regular consumption of yogurt, (7) use of any probiotic bacteria or antibiotics for one month, (8) continuous use of corticosteroids, proton pump inhibitors, statins, metformin, immunosuppressants or antitumor drugs, (9) severe cognitive impairment, hampering the performance of clinical evaluations.
37 primary tremor patients (16 of which were male patients with an average age of 62.11 ±6.88 years), 37 parkinson's disease patients (18 of which were male with an average age of 61.26 ±6.39 years) and 35 healthy controls (19 of which were male with an average age of 63.09±7.83 years) were eligible.
2. And (3) collecting clinical data: all patients in the group recorded basic demographic information, and at the same time, a unified weight scale and tape were used to measure height and weight in well-lit spaces and calculate BMI. All primary tremor patients and parkinsonian patients were diagnosed by at least two senior dyskinesia specialists and the collection of relevant clinical data was in the form of face-to-face interviews. Basic clinical data for primary tremor patients and parkinsonism patients include: age, onset of illness, course of illness, frequency and dosage of smoking, drinking alcohol, drinking tea, drinking coffee. Constipation assessment and constipation severity were using classical Roman III diagnostic criteria and the Wexner constipation scoring system, respectively. The nutritional assessment uses a food frequency scale.
3. Sample collection: all subjects in the group were collected stool samples with a unified stool sampler. All the collected fecal samples were sent to the Ruijin hospital laboratory, sub-packaged into 2ml centrifuge tubes and labeled, and stored in a-80 ℃ refrigerator for later use.
4. Screening process
Screening fecal short chain fatty acid as a primary tremor diagnosis marker can be used for differential diagnosis of parkinsonism. The specific method is shown in fig. 1, and comprises the following specific steps:
step one, detecting the content of the fecal short-chain fatty acid. For the collected fecal samples of the above subjects, the content of fecal short-chain fatty acids was detected using GC-MS, specifically as follows:
(1) 400mg of fecal sample was taken in a 2ml centrifuge tube, 50. Mu.L of 15% phosphoric acid was added, 100. Mu.L of an internal standard (isohexanoic acid) solution and 400. Mu.L of diethyl ether were further added and homogenized for 1min, and centrifuged at 12000rpm at 4℃for 10min, and the supernatant was subjected to mechanical testing.
(2) Short-chain fatty acid detection was performed using a ThermoTrace1310 gas chromatograph-mass spectrometer (GC-MS, thermoFisherScientific, USA) to extract fecal short-chain fatty acids including propionic acid, acetic acid, butyric acid, isovaleric acid, valeric acid, caproic acid, and isobutyric acid, with the following gas chromatograph-mass spectrometer conditions:
chromatographic column: using an AgilentHP-INNOWAX capillary column (30 m.times.0.25 mmID.times.0.25 um, agilent, USA); heating program: the initial temperature is maintained at 90℃for 1 minute, at 10℃per minute to 120℃followed by 5℃per minute to 150℃and finally at 25℃per minute to 250℃for 2 minutes. Carrier gas: helium, flow rate 1.0mL/min; the sample was injected at a split flow rate of 10:1 at a sample inlet temperature of 250℃and a sample volume of 1. Mu.L.
Mass spectrometry conditions: electron ionization source with electron energy of 70eV; the temperature of the gas chromatography mass spectrometry interface is 250 ℃ and the temperature of the ion source is 300 ℃; scanning mode: single ion scanning mode (SIM).
(3) Raw data generated by GC-MS were peak integrated, corrected and quantified for each fecal short chain fatty acid using MassHunter software (AgilentTech., USA), and the peak area was used to calculate metabolite concentrations.
Step two, screening differential short chain fatty acid
Obtaining specific concentration of fecal short-chain fatty acid through GC-MS, obtaining differential short-chain fatty acid according to Mann-WhitneyUtest, determining differential short-chain fatty acid propionic acid, butyric acid and isobutyric acid of primary tremor and healthy people, and determining differential short-chain fatty acid isovaleric acid and isobutyric acid of primary tremor and parkinsonism; as shown in fig. 2 (a, c, g), fecal propionic acid (861.58 ±407.74vs1128.97± 521.51ug/g; p=0.023), butyric acid (755.18 ±498.20vs1220.31±760.87; p=0.007), isobutyric acid (76.69±42.08vs106.93±60.98; p=0.040) were significantly lower than in healthy subjects; as shown in fig. 2 (d, g), fecal isovaleric acid (82.54±52.30vs104.83±75.89; p=0.014) and isobutyric acid (76.69±42.08vs113.03±54.12; p=0.005) were significantly lower than parkinson's disease in primary tremors patients;
step three, analyzing the working characteristic curve of the subject on the differential short-chain fatty acid, and determining that the fecal short-chain fatty acid is used as a primary tremor diagnosis marker and can be used for differential diagnosis of parkinsonism
From fig. 3, it can be derived that: (1) based on the data of the primary tremor patient and the healthy person, the primary tremor patient and the healthy person are judged by using propionic acid, butyric acid and isobutyric acid: the optimal cut-off for the concentration of fecal propionic acid alone to diagnose essential tremor was 675.727ug/g, at which time sensitivity was 88.6%, specificity was 45.9%, area under the curve (AUC) was 0.668 (95% ci: 0.538-0.797), less than this cut-off value, indicating that the subject had essential tremor; butyric acid alone differentially diagnoses an optimal cut-off 1502.558ug/g for primary tremor and healthy people, when sensitivity was 40.0%, specificity was 97.3%, area under the curve (AUC) was 0.685 (95% ci: 0.556-0.814), less than which cut-off value indicates that the subject had primary tremor; isobutyric acid alone differentially diagnoses an optimal cut-off of 94.236ug/g for primary tremor and healthy people, when sensitivity was 54.3%, specificity was 67.6%, area under the curve (AUC) was 0.655 (95% ci: 0.525-0.786), less than the cut-off value, indicating that the subject had primary tremor; propionic acid, butyric acid and isobutyric acid were combined to determine that the area under the curve (AUC) obtained for primary tremor and healthy persons was 0.751 (95% CI: 0.634-0.867), at which time the sensitivity was 74.3% and the specificity was 72.9%. The model for the combined differential diagnosis of essential tremors and healthy people for propionic acid, butyric acid and isobutyric acid is as follows:
Y1＝2.835-0.001*A-0.001*B-0.013*C；Prob1＝e Y1 /(1+e Y1 )；
wherein A, B, C is the concentration value of the feces obtained by the gas chromatography-mass spectrometry of propionic acid, butyric acid and isobutyric acid respectively;
prob1 represents the probability of judging essential tremor.
(2) Based on data of primary tremor patients and parkinsonian patients, isovaleric acid and isobutyric acid are used for distinguishing the primary tremor from parkinsonism: the optimal cut-off point for differential diagnosis of primary tremor and parkinson's disease alone was 79.179ug/g, with a sensitivity of 75.7%, a specificity of 54.1%, an area under the curve (AUC) of 0.700 (95% ci: 0.579-0.822), less than which cut-off value indicates that the subject had primary tremor; isobutyric acid alone differentially diagnoses an optimal cut-off 107.871ug/g for primary tremor and parkinson's disease, when sensitivity is 48.6%, specificity is 83.8%, area under the curve (AUC) is 0.718 (95% ci: 0.599-0.836), less than which cut-off value indicates that the subject has primary tremor; the area under the curve (AUC) obtained by combining isovaleric acid and isobutyric acid to discriminate primary tremor from Parkinson's disease was 0.743 (95% CI: 0.629-0.857), at which time the sensitivity was 74.3% and the specificity was 62.9%. The model for the differential diagnosis of primary tremor and parkinsonism by combining isovaleric acid and isobutyric acid is as follows:
Y2＝-2.100-0.042*A+0.067*B；Prob2＝e Y2 /(1+e Y2 )；
wherein A, B is the fecal concentration value obtained by gas chromatography-mass spectrometry of isovaleric acid and isobutyric acid, respectively.
Prob2 represents the probability of judging essential tremor.
In conclusion, the screened fecal short-chain fatty acids propionic acid, butyric acid and isobutyric acid can be used as the diagnostic markers of primary tremor, and isovaleric acid and isobutyric acid can be used for differential diagnosis of primary tremor and parkinsonism. The fecal short-chain fatty acid is used for early diagnosis of primary tremor patients and auxiliary differential diagnosis of primary tremor and parkinsonism patients, has simple operation, low cost, short detection time, high sensitivity and strong specificity, and has important clinical significance.
Implementing a secondary essential tremor diagnostic system
The embodiment provides the application of the research, and forms a primary tremor diagnosis system by the research result, and data processing and analysis are automatically realized. The diagnosis system can be arranged on a computer terminal or integrated with a gas chromatography-mass spectrometry device, and can directly analyze the concentration data of the target short-chain fatty acid and display the diagnosis result after the fecal sample is detected.
According to fig. 4, the essential tremor diagnosis system 100 of the present application includes an input display module 1, a calculation module 2, a determination module 3, a storage module 4, a communication module 5, and a control module 6.
The input display module 1 is used for inputting basic information of a patient and detection concentration of propionic acid, butyric acid, isobutyric acid and isovaleric acid in a fecal sample, or receiving target short-chain fatty acid concentration data transmitted by the gas chromatography-mass spectrometry device through the communication module and displaying a diagnosis result;
the calculation module 2 calculates the probability of suffering from essential tremor based on preset model formulas 1 and 2:
model equation 1: y1=2.835-0.001 a-0.001 b-0.013 c, prob1=e Y1 /(1+e Y1 )；
Wherein A, B, C is the concentration value of propionic acid, butyric acid and isobutyric acid detected by gas chromatography-mass spectrometry in the fecal sample; prob1 represents the probability of judging essential tremor;
model equation 2: y2= -2.100-0.042×d+0.067×e, prob2=e Y2 /(1+e Y2 )；
Wherein D, E is the concentration value of isovaleric acid and isobutyric acid obtained by gas chromatography-mass spectrometry detection in the fecal sample, and Prob2 represents the probability of judging primary tremor;
the determination module 3 determines whether the primary tremor is suffering from the primary tremor based on the following rules and transmits the result to the input display module: when the propionic acid concentration is not higher than 675.727 mug/g, the butyric acid concentration is not higher than 1502.558 mug/g, the isobutyric acid concentration is not higher than 94.236 mug/g or Prob1 is greater than 0.5, the essential tremor is judged; further, when the concentration of isovaleric acid is not higher than 79.179. Mu.g/g, the concentration of isobutyric acid is not higher than 107.871. Mu.g/g or Prob2 is more than 0.5, it is judged that essential tremor is not Parkinson's disease.
The storage module 4 is used for storing various information in the running process of the primary tremor diagnosis system; the communication module 5 is used for information transmission between different modules and information transmission between the essential tremor diagnosis system 100 and external equipment, such as communication connection between a gas chromatography-mass spectrometry device and a doctor terminal system.
The control module 6 controls the input display module 1, the calculation module 2, the judgment module 3, the storage module 4 and the communication module 5 to operate normally.
The diagnostic flow of the essential tremor diagnostic system 100 of the present application is shown in fig. 5, and includes the following steps:
s1, inputting basic information of a patient through an input display module 1, inputting or receiving detection concentrations of propionic acid, butyric acid, isobutyric acid and isovaleric acid in a fecal sample, and entering a step S2;
s2, a calculation module 2 calculates Prob1 and Prob2 based on a preset model formula 1 and a preset model formula 2, calculates the probability of suffering from primary tremors, and enters a step S3;
s3, the judging module 3 judges whether the propionic acid concentration is not higher than 675.727 mug/g, the butyric acid concentration is not higher than 1502.558 mug/g, the isobutyric acid concentration is not higher than 94.236 mug/g or Prob1 is larger than 0.5, and when the propionic acid concentration and the isobutyric acid concentration are not coincident, the process enters S6, and a diagnosis result which is not the primary tremor is output; s4 is carried out under the other conditions;
s4, the judging module 3 further judges whether the concentration of isovaleric acid is not higher than 79.179 mug/g, the concentration of isobutyric acid is not higher than 107.871 mug/g or Prob2 is larger than 0.5, when the concentrations are not coincident, the process goes to S7, the output further checks to judge whether the result is parkinsonism, and otherwise, the process goes to S5 to output the diagnosis result of primary tremor.
While the application has been described in detail with reference to the embodiments, it will be apparent to those skilled in the art that the application is not limited to the embodiments, and that the equivalent modifications and substitutions can be made without departing from the spirit of the application, and are intended to be included in the scope of the application as defined in the appended claims.
Claims (10)
1. The application of the reagent for detecting the fecal short-chain fatty acid in preparing the primary tremor diagnosis reagent or the reagent kit is characterized in that: the fecal short chain fatty acid is any one or more of propionic acid, butyric acid, isobutyric acid and isovaleric acid.
2. The use according to claim 1, characterized in that: the fecal short-chain fatty acid distinguishing the primary tremor from healthy people is any one or a combination of more of propionic acid, butyric acid and isobutyric acid; the fecal short-chain fatty acid distinguishing the primary tremor from the parkinsonism is any one or a combination of two of isobutyric acid and isovaleric acid.
3. The use according to claim 1, characterized in that:
wherein when the fecal short-chain fatty acid is propionic acid, butyric acid or isobutyric acid, the cut-off point concentration of the propionic acid as a diagnosis marker is 675.727 mug/g, the cut-off point concentration of the butyric acid as a diagnosis marker is 1502.558 mug/g, the cut-off point concentration of the isobutyric acid as a diagnosis marker is 94.236 mug/g,
the model for identifying essential tremors in healthy people is as follows:
Y1＝2.835-0.001*A-0.001*B-0.013*C，Prob1＝e Y1 /(1+e Y1 )；
wherein A, B, C is the concentration value of propionic acid, butyric acid and isobutyric acid detected by gas chromatography-mass spectrometry in the fecal sample; prob1 represents the probability of judging essential tremor;
when the fecal short-chain fatty acid is isovaleric acid and isobutyric acid, the intercept point concentration of the isovaleric acid as a diagnostic marker is 79.179 mug/g and the intercept point concentration of the isobutyric acid as a diagnostic marker is 107.871 mug/g,
the model for differential diagnosis of essential tremor and parkinson's disease is as follows:
Y2＝-2.100-0.042*D+0.067*E，Prob2＝e Y2 /(1+e Y2 )；
wherein D, E is the concentration value of isovaleric acid and isobutyric acid obtained by gas chromatography-mass spectrometry detection in the fecal sample, and Prob2 represents the probability of judging primary tremor.
4. The application of a reagent for detecting fecal short-chain fatty acid in preparing a reagent or a kit for identifying primary tremor and parkinsonism is characterized in that: the fecal short-chain fatty acid is any one or the combination of two of isobutyric acid and isovaleric acid,
the concentration of the cut-off point of the isovaleric acid as a diagnostic marker is 79.179 mug/g, the concentration of the cut-off point of the isobutyric acid as a diagnostic marker is 107.871 mug/g,
the model for differential diagnosis of essential tremor and parkinson's disease is as follows:
Y2＝-2.100-0.042*D+0.067*E，Prob2＝e Y2 /(1+e Y2 )；
wherein D, E is the concentration value of isovaleric acid and isobutyric acid detected by gas chromatography-mass spectrometry in the fecal sample, and Prob2 represents the probability of judging primary tremor.
5. Use according to claim 3 or 4, characterized in that:
wherein, the steps of detecting short chain fatty acid in the fecal sample are as follows:
(1) Taking a certain amount of fecal sample in a centrifuge tube, sequentially adding 15% phosphoric acid, 125 mug/ml internal standard isohexide solution and diethyl ether for homogenating, and centrifuging at 12000rpm at 4 ℃ for 10min to obtain supernatant;
(2) Short-chain fatty acid detection is carried out by adopting a ThermoTrace1310 gas chromatography-mass spectrometer, and target short-chain fatty acid and internal standard isocaproic acid are extracted, wherein the gas chromatography-mass spectrometer has the following conditions:
chromatographic column: an AgilentHP-INNOWAX capillary column; heating program: the initial temperature is kept at 90 ℃ for 1 minute, and is raised to 120 ℃ at 10 ℃/min, then raised to 150 ℃ at 5 ℃/min, and finally raised to 250 ℃ at 25 ℃/min for 2 minutes; carrier gas: helium, flow rate 1.0mL/min; the temperature of the sample inlet is 250 ℃, the sample is split and injected at a ratio of 10:1, and the sample injection volume is 1 mu L;
mass spectrometry conditions: electron ionization source with electron energy of 70eV; the temperature of the gas chromatography mass spectrometry interface is 250 ℃ and the temperature of the ion source is 300 ℃; scanning mode: a single ion scanning mode;
(3) Raw data generated by gas chromatography-mass spectrometry was subjected to peak integration, correction and quantitative analysis for each fecal short chain fatty acid using MassHunter software, and the metabolite concentration was calculated using peak area.
6. A primary tremor diagnostic kit comprising a fecal sample processing reagent comprising 15% phosphoric acid and diethyl ether and an internal standard reagent; the internal standard reagent is an isohexide solution of 125 mug/ml.
7. A primary tremor diagnostic system comprising:
the input display module is used for inputting basic information of a patient and detection concentration of propionic acid, butyric acid, isobutyric acid and isovaleric acid in a fecal sample, and displaying a diagnosis result;
the calculation module calculates the probability of suffering from the essential tremor based on a preset model formula 1 and a preset model formula 2:
model equation 1: y1=2.835-0.001 a-0.001 b-0.013 c, prob1=e Y1 /(1+e Y1 )；
Wherein A, B, C is the concentration value of propionic acid, butyric acid and isobutyric acid detected by gas chromatography-mass spectrometry in the fecal sample; prob1 represents the probability of judging essential tremor;
model equation 2: y2= -2.100-0.042×d+0.067×e, prob2=e Y2 /(1+e Y2 )；
Wherein D, E is the concentration value of isovaleric acid and isobutyric acid obtained by gas chromatography-mass spectrometry detection in the fecal sample, and Prob2 represents the probability of judging primary tremor;
and the judging module is used for judging whether the primary tremor is suffered from based on a preset rule and transmitting the result to the input display module.
8. The primary tremor diagnostic system of claim 7, further comprising:
the storage module is used for storing various information in the running process of the primary tremor diagnosis system;
the control module is used for controlling the input display module, the calculation module and the judgment module to normally operate;
the judging module judges based on the following rules:
when the propionic acid concentration is not higher than 675.727 mug/g, the butyric acid concentration is not higher than 1502.558 mug/g, the isobutyric acid concentration is not higher than 94.236 mug/g or Prob1 is greater than 0.5, the essential tremor is judged; further, when the concentration of isovaleric acid is not higher than 79.179. Mu.g/g, the concentration of isobutyric acid is not higher than 107.871. Mu.g/g or Prob2 is more than 0.5, it is judged that essential tremor is not Parkinson's disease.
9. A built-in electronic device in a gas chromatograph-mass spectrometer is characterized by comprising a data memory, a data processor and a computer program of a primary tremor diagnosis system which is stored on the memory and can run on the processor,
the data storage stores detection conditions of gas chromatography-mass spectrometry, raw data generated by the gas chromatography-mass spectrometry and concentration values after processing calculation, and the primary tremor diagnosis system is as set forth in any one of claims 7-8.
10. A non-transitory computer readable storage medium, on which a computer program is stored, characterized in that the computer program, when being executed by a processor, implements the steps of claim 7 or 8.
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