CN118010890B - Application of plasma amino acid in preparation of kit for evaluating or assisting in evaluating prognosis risk of hypertensive patient - Google Patents
Application of plasma amino acid in preparation of kit for evaluating or assisting in evaluating prognosis risk of hypertensive patient Download PDFInfo
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- CN118010890B CN118010890B CN202410411034.3A CN202410411034A CN118010890B CN 118010890 B CN118010890 B CN 118010890B CN 202410411034 A CN202410411034 A CN 202410411034A CN 118010890 B CN118010890 B CN 118010890B
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Abstract
The invention belongs to the field of detection methods containing amino acids, and discloses application of plasma amino acids in preparation of a kit for evaluating or assisting in evaluating prognosis risks of patients with hypertension. The invention provides a kit for evaluating or assisting in evaluating the prognosis risk of a hypertensive patient, which is characterized by comprising a sample detection unit and a judgment carrier, wherein the sample detection unit comprises a kit for detecting the content of amino acid in a biological sample of a person to be tested; and the judging carrier obtains the amino acid content according to the sample detection unit and judges the prognosis risk of the hypertension patient. The invention researches the relationship between ornithine/arginine ratio (Orn/Arg) in serum and platelet activity and prognosis condition of patients with high blood pressure and high risk, thereby evaluating prognosis risks of patients with high blood pressure.
Description
Technical Field
The invention belongs to the field of detection methods comprising amino acids, and in particular relates to application of plasma amino acids in preparation of a kit for assessing or assisting in assessing prognosis risks of patients with hypertension.
Background
Hypertension is a primary risk factor for serious cardiovascular diseases with high incidence rate, and is easy to cause various cardiovascular critical diseases including acute myocardial infarction. Although guidelines stratify cardiovascular risk in hypertensive individuals based on risk factors, target organ damage and complications, there is a great difference in event risk in individuals with the same stratification. Depending on the age, sex, dyslipidemia, and the presence of other vascular risk factors such as diabetes, the absolute cardiovascular risk of hypertensive individuals may differ by up to 20-fold at a given blood pressure level. In addition, cardiovascular risk prediction models such as guidelines are mainly used for assessing 10-year risks, and key signs of recent cardiovascular disease attacks cannot be well identified, which is very important for early warning. Thus, there is a clinical need for new plasma biomarkers to assist in identifying high-pressure, high-risk patients who actually develop adverse cardiovascular events.
Disclosure of Invention
The technical problem to be solved by the invention is how to prepare a product for predicting prognosis of a patient with high blood pressure and high risk and/or how to obtain a marker for predicting prognosis of a patient with high blood pressure and high risk.
In order to solve the above technical problems, the present invention provides a kit for assessing or assisting in assessing the prognosis risk of a patient suffering from hypertension, comprising a sample detection unit and a judgment carrier, wherein the sample detection unit comprises a substance for detecting at least one of the following 27 types of amino acids in a biological sample of a person to be tested, the 27 types of amino acids are lysine, histidine, glycine, threonine, asparagine, aspartic acid, alanine, glutamic acid, glutamine, citrulline, ornithine, urea, proline, creatine, leucine, isoleucine, valine, methionine, tyrosine, phenylalanine, tryptophan, kynurenine, serine, arginine, 4-hydroxyproline and taurine, respectively; and the judging carrier obtains the amino acid content according to the sample detection unit and judges the prognosis risk of the hypertension patient.
As a further scheme, the sample detection unit comprises a substance for detecting the content of ornithine and arginine in the biological sample of the person to be detected, and the judgment carrier is used for calculating the ornithine/arginine ratio according to the ornithine and arginine content obtained by the sample detection unit and judging the prognosis risk of the patient with hypertension.
As a further scheme, the prognosis risk of the hypertension patient refers to the risk of adverse events of the hypertension patient, the judgment carrier obtains the amino acid content according to the sample detection unit, and the criterion for judging the risk of adverse events of the hypertension patient is as follows: in biological samples of the testee, patients with hypertension, with plasma Orn/Arg ratio higher than 0.66, have a high risk of adverse events during hospitalization.
As a further approach, the risk of cardiovascular events (MACE) for the high-pressure, high-risk patient refers to the risk of cardiovascular death, non-lethal myocardial infarction, heart failure in hospitalization due to heart failure attacks, ischemia-driven urgent revascularization, transient ischemic attacks, ischemic stroke or hemorrhagic stroke for the high-pressure, high-risk patient.
As a further scheme, the judging carrier obtains the amino acid content according to the sample detection unit, and the standard for judging the prognosis risk of the hypertension patient is as follows: in biological samples of subjects, plasma Orn/Arg ratios below 0.66 are better for prognosis in high-pressure, high-risk patients.
As a further scheme, the prognosis risk of the hypertension patient refers to the platelet activity condition of the hypertension high-risk patient, the judgment carrier obtains the amino acid content according to the sample detection unit, and the standard for judging the platelet activity condition of the hypertension high-risk patient is as follows: the plasma Orn/Arg ratio is strongly correlated with platelet activity, i.e. the higher the plasma Orn/Arg ratio the higher the platelet activity.
As a further aspect, the biological sample is a blood sample, a serum sample, or a plasma sample; the substance for detecting the amino acid content in the biological sample of the person to be detected comprises reagents and devices required for extracting the amino acid in the biological sample and measuring by using LC-MS/MS.
The use of a substance for detecting at least one of the following 27 classes of amino acids in a biological sample of a subject for the preparation of any of the following kits shall also fall within the scope of the present invention:
(a1) Kits for screening or aiding in diagnosing patients with hypertension;
(a2) A kit for assessing or aiding in assessing the risk of prognosis of a hypertensive patient;
(a3) A kit for assessing or aiding in assessing the risk of an adverse event in a hypertensive patient.
The invention provides a kit for screening or assisting in screening patients with hypertension, which comprises a sample detection unit, a standard substance and a judging carrier, wherein the sample detection unit comprises a substance for detecting at least one of 27 types of amino acids in a biological sample of a person to be tested, wherein the 27 types of amino acids are lysine, histidine, glycine, threonine, asparagine, aspartic acid, alanine, glutamic acid, glutamine, citrulline, ornithine, urea, proline, creatine, leucine, isoleucine, valine, methionine, tyrosine, phenylalanine, tryptophan, kynurenine, serine, arginine, 4-hydroxyproline and taurine respectively; the judging carrier judges whether the person to be detected is or is a candidate hypertension patient according to the difference between the amino acid content obtained by the sample detecting unit and the standard substance; the standard substance is the content of lysine, histidine, glycine, threonine, asparagine, aspartic acid, alanine, glutamic acid, glutamine, citrulline, ornithine, urea, proline, creatine, leucine, isoleucine, valine, methionine, tyrosine, phenylalanine, tryptophan, kynurenine, serine, arginine, 4-hydroxyproline and taurine in biological samples of healthy people.
As a further option, the screening or auxiliary screening kit is used for screening patients with hypertension who are at different risk for prognosis.
As a further aspect, the biological sample is a blood sample, a serum sample, a plasma sample; the substance for detecting the amino acid content in the biological sample of the person to be detected comprises reagents and devices required for extracting the amino acid in the biological sample and measuring by using LC-MS/MS.
As a further scheme, the sample detection unit comprises a substance for detecting the content of ornithine and arginine in the biological sample of the person to be detected, and the judgment carrier is used for calculating the ornithine/arginine ratio according to the ornithine and arginine content obtained by the sample detection unit and judging the patients with hypertension with different prognosis risks.
As a further scheme, the prognosis risk of the hypertension patient refers to the risk of adverse events of the hypertension patient, the judgment carrier obtains the amino acid content according to the sample detection unit, and the judgment standard is as follows: among the biological samples of the subjects, the patients with hypertension having a plasma Orn/Arg ratio higher than 0.66 are patients with hypertension at high risk of adverse events during hospitalization.
From the aspect of guiding clinical prevention strategies for patients with high blood pressure and high risk, the ornithine/arginine ratio is found to be used as a marker for occurrence and development of hypertension by detecting the amino acid expression level of the patients with high blood pressure and high risk, and is possibly used for prompting platelet activity and is independently related to occurrence of recent adverse events of the patients with high blood pressure and high risk.
Based on the problems in the prior clinic, the relationship between ornithine/arginine ratio (Orn/Arg) in serum and platelet activity and prognosis of patients with high blood pressure and high risk is studied, 51 patients with high blood pressure and high risk of MACEs and 562 patients with high blood pressure and high risk of Non-MACEs are retrospectively collected, the amino acid level in the serum is quantified by using an LC-MS/MS technology, and a statistical result shows that the ornithine/arginine ratio can be used as a prediction index of adverse cardiovascular events of the patients with high blood pressure and high risk, and compared with the existing clinical detection index, the ornithine/arginine ratio can better and effectively distinguish the patients with high blood pressure and high risk of recent adverse events.
The invention utilizes the LC-MS/MS technology to quantify the amino acid level in serum of patients with high blood pressure and high risk, finds that the ornithine/arginine ratio of the serum is related to the higher platelet activity of the patients with high blood pressure and high risk, can be used as a prediction index of the recent adverse cardiovascular events of the patients, and can be used for preparing a product for predicting or assisting the prediction of the patients with high blood pressure and high risk. The prognosis of patients with high blood pressure and high risk can be predicted by detecting the ornithine and arginine content in serum, and the prognosis of patients with high blood pressure and high risk with the ratio of plasma Orn/Arg lower than 0.66 is better than that of patients with high blood pressure and high risk with the ratio of plasma Orn/Arg higher than 0.66, and the ratio of plasma Orn/Arg is strongly related to the platelet activity, namely, the higher the ratio of plasma Orn/Arg is, the higher the platelet activity is. The invention can improve the risk prediction capability of MACEs patients with high blood pressure and high risk on the basis of known risk factors, and is used for accurately layering the risks of the patients in clinic, thereby improving the survival rate of the patients.
Drawings
Fig. 1 is a comparison of levels of arginine and ornithine in plasma of a healthy control group and a hypertensive group, wherein the left graph is a comparison of levels of arginine in plasma of a healthy control group and a hypertensive group, and the right graph is a comparison of levels of ornithine in plasma of a healthy control group and a hypertensive group, wherein the graph indicates a statistically significant difference in amino acid levels between the two groups (P-value less than 0.0001).
Figure 2 is a comparison of the risk capacity of different COX regression models for evaluating high blood pressure and high risk patients MACEs at a statistical amino acid level, wherein the left panel shows the risk capacity of COX regression models for evaluating high blood pressure and high risk patients MACEs senior sister apprentice at a statistical amino acid level in a development queue, and the right panel shows the risk capacity of COX regression models for evaluating high blood pressure and high risk patients MACEs at a statistical amino acid level in a validation queue, with the horizontal line and the square block representing the 95% confidence interval and HR, respectively, for two different COX regression models.
FIG. 3 is a graph of the subject work profile of Orn/Arg versus high risk for high blood pressure MACEs and Non-MAEs patients, where the left graph is the subject work profile of Orn/Arg versus high risk for high blood pressure MACEs and Non-MACEs patients in the development cohort and the right graph is the subject work profile of Orn/Arg versus high risk for high blood pressure MACEs and Non-MACEs patients in the validation cohort.
FIG. 4 shows the results of the measurement of the Orn/Arg ratio and the platelet activity in the plasma of patients with high risk of hypertension, wherein the left graph shows the difference between the Orn/Arg ratio of patients with high risk of hypertension MACEs and Non-MACEs, the right graph shows the relationship between the Orn/Arg ratio and the platelet activity of patients with high risk of hypertension MACEs and Non-MACEs, the triangle in the scatter diagram represents Non-MACEs, the circle represents MACEs, and the graph shows that the statistical difference exists between the two groups (P value is less than 0.05).
FIG. 5 shows the correlation of the plasma Ogn/Arg ratio with platelet activity in patients at high risk of hypertension.
FIG. 6 is a schematic illustration of the formulation of the internal standard solution according to the examples.
FIG. 7 is a comparison of plasma amino acid levels in healthy and hypertensive patients.
FIG. 8 is a comparison of plasma amino acid levels in patients with hypertensive and non-incident groups.
FIG. 9 is a correlation of ornithine/arginine ratio in plasma of high blood pressure and high risk patients with MACEs.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The study in the examples below is a cross-sectional study, the study population being primarily from a cardiovascular event Prognosis Risk (PROSPECT) registration study (NCT 03708601) for primary hypertension patients. First, collect 2016, 9 to 2018, 10 and visit Beijing An Zhen hospital, and the patients with primary hypertension aged 18-85 years. Exclusion criteria: hypertension caused by secondary causes; evidence of angina attacks, myocardial infarction, or PCI or CABG in the first 3 months; stroke or other ischemic cerebrovascular episodes (cerebral infarction, cerebral hemorrhage, TIA) within the first 3 months; serious valvular disease, congestive heart failure (NYHAII-grade IV) or LVEF < 40%; severe liver disease; severe renal failure (defined as CREA. Gtoreq.265 umol/L); combining pregnancy and tumor; patients with loss of access. Among them, the diagnosis of cardiovascular high risk or extremely high risk is determined according to the "China guidelines for hypertension control (revised in 2018)". The diagnostic criteria for high and extremely high cardiovascular risk are any of the following: ① Any of the following diseases: coronary heart disease, congestive heart failure, atrial fibrillation, cerebrovascular disease or peripheral arterial occlusive disease, diabetes (the only missing variable is information on retinopathy for the complications mentioned in the guidelines), ② any form of organ damage, including men with a left ventricular mass index of ≡115 g/m 2, The female is more than or equal to 95 g/m < 2 >, the carotid intima-media thickness is more than or equal to 0.9 mm or carotid plaque, the pulse wave velocity is more than or equal to 12 m/s, the brachial ankle index is less than or equal to 0.9, the male serum creatinine is more than or equal to 115 [ mu ] mol/l and the female is more than or equal to 107 [ mu ] mol/l, or the urinary albumin creatinine ratio is more than or equal to 30 mg/g, the ③ grade 1 or grade 2 high blood pressure is accompanied by at least 3 other CV risk factors, including males with ages of more than or equal to 55 years and females with ages of more than or equal to 65, Smoking (current smoking or past smoking history), abnormal glucose tolerance (fasting blood glucose 6.1-6.9 mmol/l), low density lipoprotein cholesterol not less than 4.1 mmol/l or high density lipoprotein cholesterol not less than 1.0 mmol/l, early onset CVD family history, body mass index not less than 28 kg/m 2, plasma homocysteine not less than 15 [ mu ] mol/l, or ④ hypertension grade 3. the risk of cardiovascular events (MACE) in high-pressure, high-risk patients refers to the risk of cardiovascular death, non-fatal myocardial infarction, hospitalized heart failure due to heart failure attacks, ischemia-driven urgent revascularization, transient ischemic attacks, ischemic strokes, or hemorrhagic strokes in the high-pressure, high-risk patients.
The detection of 27 amino acid contents in plasma in the following examples means that the 27 amino acid contents in plasma were detected and quantified by using Thermo Q Exactive mass spectrometer. The 27 amino acids are lysine, histidine, glycine, threonine, asparagine, aspartic acid, alanine, glutamic acid, glutamine, citrulline, ornithine, urea, proline, creatinine, creatine, leucine, isoleucine, valine, methionine, tyrosine, phenylalanine, tryptophan, kynurenine, serine, arginine, 4-hydroxyproline, taurine.
The test samples in the following examples: the laboratory test sample is blood plasma, 3-5mL venous blood of a tested person is extracted, the blood collection tube is injected, the blood collection tube is gently inverted immediately and mixed for 5-10 times, so that the anticoagulant is fully and uniformly mixed with the venous blood, and the test is carried out in a sealed mode. The specimens are preferably used for testing immediately or stored at-80℃to avoid repeated freeze thawing.
The main experimental procedure involved in the following examples:
1. The formulation of the internal standard solution is shown in figure 6. Wherein all the above standards were dissolved with 30% aqueous methanol. The internal standard solution is diluted by 50 times with water to be used as an internal standard working solution.
2. Sample pretreatment: the samples were removed from the-80 ℃ refrigerator and thawed in a 4 degree refrigerator. 50uL of each sample was taken, 200uL of glacial acetonitrile was added, and vortexed at 4 degrees for 30 minutes. Centrifuge at 4℃14000 RPM for ten minutes and remove 100uL. Nitrogen blowing until blow-drying. Redissolving with 100uL internal standard working solution. After vortexing at room temperature for 1 minute, centrifugation was carried out for 5 minutes at a rotational speed of 4℃14000 RPM. 20uL of the sample is filled into a sample bottle. 2uL of sample was introduced.
3. Chromatographic column treatment: when the chromatographic column is replaced, for amino acid detection, 90% of solution B and 10% of solution A are firstly used for washing for 30min, and then 50% of solution B and 50% of solution A are used for washing for 30min (A is aqueous solution containing 0.1% of formic acid; B is acetonitrile).
4. Chromatographic conditions: the amino acid metabolite profiles of plasma samples were performed using Ultimate 3000 UHPLC ultra high performance liquid chromatography (Thermo Scientific, san Jose, calif., USA) with Q Exactive HFMS mass spectrometry detection systems (Thermo Scientific, sanJose, CA, USA). The chromatograph sets the sample volume to 10 μl. The temperature of the chromatographic column is 40 ℃; the flow rate was 0.3 mL/min. In the positive ion mode, a HESST column (100 mm × 2.1 mm,1.8 mm;Waters,Milford,MA,USA) was used. The mobile phases A and B were water and acetonitrile (each containing 0.1% v/v formic acid and 5mM ammonium acetate), respectively. The elution gradient is: the initial mobile phase was 2% B, the mobile phase was 20% B at 7min, 50% B at 10min, to 98% at 10.1min, then held at this state for 4min, the mobile phase was restored to 2% B at 14.1 min, and the mobile phase ratio was held to 17min gradient end.
5. Mass spectrometry conditions: the ion spray voltage in positive ion mode was 4.5kV using a heated electrospray ionization source (ESI). The capillary temperature and heater temperature were set at 350 ℃. The collision gas was argon, a pressure of 1.5mTorr, and a collision energy of 1.5V. Sheath gas flow rate was 30psi and auxiliary gas flow rate was 10psi. Data were collected using a selective reaction monitoring scan mode (SRM). Off-press data was imported into Xcalibur 2.5 software (Thermo Scientific, san Jose, CA, USA) for data preprocessing such as baseline filtering, peak identification, integration, retention time correction, peak alignment and normalization for further analysis. The content of the corresponding amino acid in each sample is calculated by the ratio of the quantitative internal standard peak area of each amino acid to the peak area of the amino acid in the sample.
6. Platelet activation detection specific experimental method: activated platelets in circulation (namely, the surface simultaneously expresses a platelet marker CD41 and an activated marker CD 62P) are detected by using flow cytometry, so that the activation degree and the functional state of the platelets in the body can be intuitively, sensitively and specifically reflected. The peripheral blood of the patient was collected by using a sodium citrate anticoagulation blood collection tube and subjected to low-speed centrifugation at 100 Xg at room temperature for 10 minutes (acceleration set to 4 and deceleration set to 0) to obtain Platelet Rich Plasma (PRP). Platelet rich plasma was diluted in PBS at a dilution ratio of 1:50, fluorescein Isothiocyanate (FITC) -conjugated anti-CD 41 monoclonal antibody (mAb CD41-FITC; BD Bioscience, san Jose, calif., USA) was added, and Phycoerythrin (PE) -conjugated anti-CD 62P monoclonal antibody (mAb CD62P-PE; BD Bioscience, san Jose, calif., USA) was incubated for 30min in the absence of light, and 2% paraformaldehyde was added for immobilization. At least 10,000 events per sample were collected using BD LSRFortessa (BD Bioscience) flow cytometer with BD FACSDiva software (BD Bioscience) to analyze resting (cd41+) and activated (cd41+/cd62p+) platelets.
The system for detecting the amino acid level is applied to preparation of a product for predicting or assisting in predicting a high-risk patient with high blood pressure. The kit comprises a product for detecting the content of amino acids such as arginine, ornithine and the like in biological samples of patients with high risk of hypertension. The biological sample is a blood sample, a serum sample, a plasma sample or a blood lipid fraction or a blood lipoprotein fraction obtained therefrom; the product for detecting the amino acid content in the biological sample of the high-pressure high-risk patient comprises reagents required for extracting the amino acid in the biological sample and measuring by using a liquid chromatography-mass spectrometry (LC-MS/MS) technology. The amino acids include the following 27 classes: lysine, histidine, glycine, threonine, asparagine, aspartic acid, alanine, glutamic acid, glutamine, citrulline, ornithine, urea, proline, creatine, leucine, isoleucine, valine, methionine, tyrosine, phenylalanine, tryptophan, kynurenine, serine, arginine, 4-hydroxyproline, taurine.
The diagnosis method comprises the following steps: lysine, histidine, glycine, threonine, asparagine, aspartic acid, alanine, glutamic acid, glutamine, citrulline, ornithine, urea, proline, creatine, leucine, isoleucine, valine, methionine, tyrosine, phenylalanine, tryptophan, kynurenine, serine, arginine, 4-hydroxyproline and taurine in a biological sample of a patient at high risk of hypertension to be measured. Analysis of amino acid levels and ornithine/arginine ratios as a function of MACEs and ischemic events and platelet activity by COX regression models; ornithine/arginine ratios > 0.66 are considered to have a high risk of cardiovascular events and a poor prognosis. The application method of the kit comprises the following steps:
1) Detecting the amino acid content in a biological sample of a patient with high blood pressure and high risk;
2) Determining the concentration of amino acid in the sample of the high-pressure high-risk patient;
3) For the patient to be tested, when the ornithine/arginine ratio in the biological sample is > 0.66, higher platelet activity and risk of cardiovascular adverse events, worse prognosis are considered.
Example 1: the amino acid levels of the healthy control group and the hypertensive group patients were compared.
For matching according to gender age tendency scores (using nearest neighbor matching method, calipers value set to 0.02), 24 healthy controls (healthy control group) and 22 hypertensive patients (hypertensive group) were included, and plasma 27 amino acid levels of the patients were detected by mass spectrometry. The results are shown in the tables in fig. 1 and 7.
As can be seen from the results, the concentrations of lysine, histidine, glycine, threonine, asparagine, aspartic acid, alanine, glutamic acid, glutamine, citrulline, ornithine, urea, proline, creatinine, creatine, leucine, isoleucine, valine, tyrosine, phenylalanine, tryptophan, kynurenine are significantly increased (P < 0.05), and the concentrations of serine, arginine, taurine are significantly decreased (P < 0.05) in the patients of the hypertension group compared with the patients of the healthy control group.
EXAMPLE 2 prognostic evaluation value of ornithine/arginine ratio in plasma for adverse cardiovascular events (MACEs) in patients at high risk for hypertension
1. Amino acid levels were compared in the hypertensive group MACEs and the Non-MACEs groups.
The development cohort 584 and the 178 high-pressure high-risk patients of the validation cohort were followed for 3 years, and the high-pressure high-risk patients in example 1 were classified into an event group (MACEs event occurred during the follow-up period) and a non-event group (i.e., MACEs event occurred during the follow-up period) according to the occurrence of MACEs events during the follow-up period, and the plasma 27 amino acid levels of the patients were detected by mass spectrometry.
As shown by the data in fig. 8, the concentrations of lysine and ornithine in the event group patients were significantly elevated (P < 0.05) and the concentrations of arginine were significantly reduced (P < 0.05) consistently in both the development and validation cohorts as compared to the non-event group patients.
In combination with the results in fig. 1, ornithine was elevated in patients with hypertension and the level of patients with hypertension with cardiovascular adverse events was higher, as was arginine reduced in patients with hypertension and further reduced in patients with cardiovascular adverse events.
2 The relationship between amino acid level and MACEs was analyzed by Cox regression model.
Plasma amino acid testing was performed in 584 high-blood pressure, high-risk patients in the development cohort and 178 high-blood pressure, high-risk patients in the validation cohort in step 1. Two Cox regression models were developed to evaluate the evaluation value of plasma amino acid levels for high-pressure, high-risk patients MACEs and ischemic events according to cardiovascular risk factors of the hypertension guidelines. The specific building method of the model is as follows:
in model 1 (unadjusted uncorrected model), the risk assessment capacity of each amino acid for MACEs events was analyzed with the amino acid alone as an independent variable, MACEs as a dependent variable, respectively, without correcting any known risk factors;
In model 2 (adjusted correction model), the risk factors set forth in the hypertension guidelines, including hypertension grade, age, sex, smoking, family history of early cardiovascular disease, body mass index, fasting blood glucose, total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, homocysteine, left ventricular mass index, carotid intima-media thickness ≡0.9mm or carotid plaque, pulse wave velocity, brachial ankle index, plasma creatinine, urinary albumin-creatinine ratio +30 mg/g, coronary artery disease, atrial fibrillation, cerebrovascular disease, peripheral arterial disease and diabetes were also included with each amino acid as independent variables to evaluate the independent risk assessment ability of each amino acid for MACEs events after correction of patient characteristics;
As a result, as shown in fig. 2, it can be seen from fig. 2 that arginine and ornithine can be independently evaluated for the risk of high blood pressure, high risk patients MACEs and ischemic events after correction of patient characteristics.
3 The relationship between ornithine/arginine ratio (Orn/Arg) and MACEs events was analyzed by COX regression model.
Plasma amino acid tests were performed on 584 high-blood pressure and high-risk patients in the development cohort and 178 high-blood pressure and high-risk patients in the validation cohort in step 1, and the ornithine-to-arginine ratio (Orn/Arg) was calculated based on the arginine and ornithine contents in the plasma. Two Cox regression models were developed to evaluate the value of plasma ornithine/arginine ratios for high blood pressure, high risk patients MACEs and ischemic events, based on cardiovascular risk factors in the hypertension guidelines.
As shown in the data in FIG. 9, the ratio of Orn/Arg to MACEs risk was 2.11 (95% confidence interval: 1.75-2.53, P < 0.001) in model 1 without correction of any factors in the development queue; after model 2 corrects for patient risk factors, the ratio of Orn/Arg to MACEs risk is 2.16 (95% confidence interval: 1.59-2.93, P < 0.001). In the validation queue, the ratio of Orn/Arg to MACEs risk was 2.11 (95% confidence interval: 1.75-2.53, P < 0.001) when model 1 did not correct any factor; after model 2 corrects for patient risk factors, the ratio of Orn/Arg to MACEs risk is 2.16 (95% confidence interval: 1.59-2.93, P < 0.001). The above results indicate that Orn/Arg has an evaluation capacity for MACEs risk and that MACEs risk can still be predicted independently after correction of known risk factors.
Further, a subject working characteristic curve of Orn/Arg for distinguishing high-pressure high-risk patients from non-event patients is drawn, as shown in FIG. 3, by using the subject working characteristic curve, the critical value (risk value) of Orn/Arg is determined to be 0.66 in a development queue, and the area under the curve is 0.75, so that a high-pressure high-risk patient with MACEs events and a patient without MACEs events can be effectively distinguished, namely, a high-pressure high-risk patient with a plasma Orn/Arg ratio higher than 0.66 has a risk of MACEs events higher than a high-pressure high-risk patient with a plasma Orn/Arg ratio lower than 0.66 within 3 years after discharge; high-pressure, high-risk patients with a plasma Orn/Arg ratio below 0.66 have a better prognosis than those with a plasma Orn/Arg ratio above 0.66. The predicted efficacy of the plasma Orn/Arg ratio was verified in the verification queue, with an area under the curve of 0.72, which effectively distinguished between high-pressure, high-risk patients with MACEs events and those without MACEs events.
Example 3 ornithine/arginine ratio in plasma of patients at high risk for high blood pressure was related to platelet activation.
19 Patients at high blood pressure and high risk with MACEs and 43 patients at high blood pressure and high risk with no-MACEs were collected as studies of the relationship between ornithine/arginine ratio and platelet activation in plasma of patients at high blood pressure and high risk. Plasma arginine and ornithine levels were measured in patients by mass spectrometry and the Orn/Arg ratio was calculated. Platelet activity was assessed by flow cytometry for platelet marker CD41a and platelet activation marker CD62P and correlation analysis was performed with their plasma Orn/Arg ratio.
The results of the plasma Orn/Arg ratio test are shown in FIG. 4, and the plasma Orn/Arg ratio of the high-pressure and high-risk patients in the non-event group is significantly increased compared with that of the high-pressure and high-risk patients in the event group.
The correlation of plasma Orn/Arg ratio with platelet activity was analyzed by Spearman and the results are shown in FIG. 5. The Spearman correlation coefficient was 0.33 (p=0.008), indicating that the plasma Orn/Arg ratio correlated strongly with platelet activity, i.e. the higher the plasma Orn/Arg ratio, the higher the platelet activity.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (2)
1. The application of ornithine and arginine in preparing a kit for evaluating or assisting in evaluating the prognosis risk of a hypertensive patient is characterized in that the content of ornithine and arginine in a biological sample of a person to be tested is detected through a sample detection unit, the ornithine/arginine ratio is calculated, and the prognosis risk of the hypertensive patient is judged; the prognosis risk of the hypertension patient refers to the risk of the hypertension patient for cardiovascular events, and the standard for judging the risk of the hypertension patient for cardiovascular events is as follows: in the biological sample of the person to be tested, the risk of cardiovascular events of the hypertensive patient with ornithine/arginine ratio higher than 0.66 is high; the risk of cardiovascular events of the hypertensive patient refers to the risk of cardiovascular death, non-lethal myocardial infarction, heart failure in hospitalization due to heart failure attacks, ischemia-driven urgent revascularization, transient ischemic attacks, ischemic stroke or hemorrhagic stroke of the hypertensive patient; the biological sample is a blood sample, a serum sample or a plasma sample.
2. The use according to claim 1, wherein the criterion for determining the prognosis risk of a hypertensive patient on the basis of the ornithine and arginine contents obtained by the sample detection unit is: in the biological sample of the person to be tested, the risk of cardiovascular events is low for patients with hypertension having ornithine/arginine ratios below 0.66.
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| 血浆游离氨基酸的水平变化与高血压病的相关性;刘春兰, 刘勇, 曲卫, 柳青, 毕春园, 许建鸿;实用医药杂志;20021230(第12期) * |
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