CN114646702B

CN114646702B - Application of cholesterol sulfate detection reagent in preparation of sepsis auxiliary diagnosis, treatment effect monitoring and prognosis evaluation kit

Info

Publication number: CN114646702B
Application number: CN202210211153.5A
Authority: CN
Inventors: 苏智广; 于晓倩; 刘银; 张红梅
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2022-03-03
Filing date: 2022-03-03
Publication date: 2023-01-06
Anticipated expiration: 2042-03-03
Also published as: CN114646702A

Abstract

The invention provides an application of a cholesterol sulfate detection reagent in preparation of a sepsis auxiliary diagnosis, treatment effect monitoring and prognosis evaluation kit, belonging to the field of biomarkers. The invention discovers that the content of cholesterol sulfate in early serum of a sepsis patient is obviously increased through research, and the content of the cholesterol sulfate is reduced when the symptoms are aggravated and enter a shock state, so that the cholesterol sulfate can be used as an auxiliary index for evaluating the severity of sepsis. In addition, the treatment effect of the sepsis can be monitored in an auxiliary way and the prognosis evaluation can be carried out according to the expression level of cholesterol sulfate during the treatment process. The kit is used for auxiliary diagnosis, auxiliary monitoring of treatment effect and auxiliary prognosis evaluation of sepsis by detecting cholesterol sulfate in serum, provides a method for auxiliary monitoring of clinical sepsis, provides effective basis for patients to take relevant treatment measures or decisions, and has good clinical application prospect.

Description

Application of cholesterol sulfate detection reagent in preparation of sepsis auxiliary diagnosis, treatment effect monitoring and prognosis evaluation kit

Technical Field

The invention belongs to the field of biomarkers, and particularly relates to an application of a cholesterol sulfate detection reagent in preparation of a sepsis auxiliary diagnosis, treatment effect monitoring and prognosis evaluation kit.

Background

Sepsis is a systemic inflammatory response syndrome caused by infection that can progress to septic shock and multiple organ dysfunction syndrome, which can be life threatening. Activation of inflammatory cells and production of large amounts of inflammatory mediators lead to damage, dysfunction and even failure of multiple organs during the onset of sepsis. With the intensive research on the pathogenesis and mechanism of sepsis in recent years, the concept of sepsis has been updated to sepsis3.0, and various treatment technologies such as 1h cluster therapy, antibiotic management, immunoregulation, organ support and the like have been advanced. At present, the incidence rate of sepsis in patients admitted to ICU in China is still as high as 37.3%, the fatality rate is 28.7%, and the life quality of patients suffering from sepsis is reduced compared with that before illness. In terms of the long term mortality, the 1-year mortality of patients with sepsis is 23%, the 2-year mortality is 28.8%, and the 5-year mortality reaches 43.8%. Therefore, how to accurately diagnose the sepsis, how to effectively monitor the treatment effect in the treatment process and if accurately carrying out prognosis evaluation on the sepsis are of great significance to patients with the sepsis.

For the diagnosis of sepsis, blood-related examinations, etiology examinations and imaging examinations are mainly combined clinically. The anti-inflammatory treatment of sepsis has made a series of progress, but still faces many challenges, the curative effect and safety of anti-inflammatory and immune target therapy are unknown, and because of the problems of patient gene difference, different immune cells and cell signal path networks when sepsis occurs, the interaction of different cells and organs, whether all current treatment schemes interfere the adaptation process of the organism to sepsis, selection of proper treatment time and improvement of clearance specificity, close connection between gene polymorphism of various inflammatory mediators and sepsis, how to select individualized treatment mode for patients, and the like, further research is needed. If a biomarker can be researched, sepsis can be diagnosed in an auxiliary way, occurrence and development of diseases are identified according to the change of the content of the biomarker, the treatment effect is monitored in an auxiliary way in the treatment process, the prognosis evaluation is assisted, auxiliary guidance is performed in the sepsis diagnosis, treatment and prognosis processes, and the method has important significance for sepsis treatment.

At present, sepsis biomarkers for sepsis diagnosis, monitoring of therapeutic effect, and prognosis evaluation mainly include C-reactive protein (CRP) and Procalcitonin (PCT). CRP can be combined with capsular C polysaccharide of streptococcus pneumoniae, is generated by stimulation of inflammatory factors such as IL-6 and the like, is mainly increased 4-6 hours after the inflammatory response of infectious or non-infectious contact, reaches a peak 36-50 hours, and is reduced along with the regression of inflammation. PCT is the propeptide material of serum calcitonin, produced by secretion from thyroid C cells. Under the condition of inflammatory stimulation, various cells of various tissues of an organism can generate PCT and release the PCT into blood, the peak can be reached in 24-36 hours, and the PCT has stronger specificity on bacterial infection. Both CRP and PCT are associated with bacterial infections, fail to truly reflect the functional status of individual organs and the state of nutritional energy metabolism of the body, and are deficient in both sensitivity and specificity. Therefore, the search for novel biomarkers is of great significance in the application of auxiliary diagnosis, treatment monitoring and prognosis evaluation of clinical sepsis and the nutritional energy state of the organism.

Cholesterol sulfate sodium salt (CS) is a main steroid sulfate in human serum, has high content, is transported in combination with LDL, is formed by replacing a hydrogen atom at the 3 rd position of a Cholesterol molecule by a sulfoxy group, and has the average level of CS in female serum lower than that in male serum. The synthesis and hydrolysis of CS is accomplished by steroid Sulfotransferase (SULT) and steroid sulfatase (STS), respectively. In recent years, research shows that CS can be used as a signal molecule and has important functions of regulating cellular oxidative stress, glycolipid metabolism, immunity and survival. CS can induce mRNA transcription of antioxidant enzyme, superoxide dismutase 2 and GPx1 by activating endogenous ligand ROR alpha, inhibit ROS generation, and regulate apoptosis. The sensitivity of the mouse to drug-induced liver injury can be enhanced by over-expressing the CS synthetase encoding gene Sult2b1b in the mouse liver.

At present, the role of CS in sepsis is not completely clear, and the change of the CS content in serum in the role of sepsis occurrence and development and the role thereof have not been reported at home and abroad.

Disclosure of Invention

The invention aims to provide a novel biomarker cholesterol sulfate, and particularly provides application of a cholesterol sulfate detection reagent in preparation of a sepsis auxiliary diagnosis, auxiliary treatment effect monitoring and auxiliary prognosis evaluation kit.

The invention provides application of a reagent for detecting cholesterol sulfate in preparation of a kit for sepsis auxiliary diagnosis, treatment effect auxiliary monitoring and/or auxiliary prognosis evaluation.

Further, the reagent for detecting cholesterol sulfate is a reagent for detecting the content of cholesterol sulfate in human blood.

Further, the reagent for detecting cholesterol sulfate is a reagent for detecting the content of cholesterol sulfate in human serum.

Furthermore, the detection method of the reagent for detecting cholesterol sulfate adopts liquid chromatography tandem mass spectrometry detection.

Further, the chromatographic detection conditions were as follows:

preferably, the elution mode of the chromatographic detection is gradient elution, and the elution conditions are as follows:

further, the mass spectrometric detection conditions are as follows:

electrospray ionization source, negative ion scanning mode, GAS1:60psi, GAS2:60psi, curtainGAS:20psi, source temperature 500 ℃, ion spray voltage (IS) 4500V, declustering voltage (DP) 100V, ion pair of CS 465.1-96.9; the collision energy was 43eV, the ion pair for the internal standard was 265.1-223.1, and the collision energy was 45eV.

The invention also provides a sepsis auxiliary diagnosis, treatment effect auxiliary monitoring and/or auxiliary prognosis evaluation kit, which comprises the reagent for detecting cholesterol sulfate;

preferably, the kit comprises the following components: (1) an aqueous solution containing 0.01% ammonia water; (2) acetonitrile; and (3) a magnolol standard substance.

Further, the chromatographic detection conditions were as follows:

further, the mass spectrometric detection conditions are as follows:

In the present invention, the sodium salt of cholesterol sulfate is referred to as cholesterol sulfate.

The research shows that the cholesterol sulfate can be used as an index for auxiliary diagnosis, auxiliary monitoring and auxiliary prognosis evaluation of sepsis: the content of cholesterol sulfate in early serum of patients with sepsis is obviously increased, and the patients enter a shock state along with aggravation of symptoms, so that the content of the cholesterol sulfate is reduced, and the cholesterol sulfate can be used as an auxiliary index for evaluating the severity of the sepsis. In addition, the treatment effect of the sepsis can be monitored in an auxiliary way and the prognosis evaluation can be carried out according to the expression level of cholesterol sulfate during the treatment process. The kit is used for auxiliary diagnosis, auxiliary monitoring of treatment effect and auxiliary prognosis evaluation of sepsis by detecting cholesterol sulfate in serum, provides a method for auxiliary monitoring of clinical sepsis, provides effective basis for patients to take relevant treatment measures or decisions, and has good clinical application prospect.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

Figure 1 shows the SOFA score criteria.

FIG. 2 is a standard curve of CS.

FIG. 3 shows the time to peak for the internal standard and standards: a is the internal standard peak-producing time, and b is the standard peak-producing time.

Figure 4 is data statistics for sepsis patients and healthy examiners.

Fig. 5 shows the CS content in serum of sepsis patients and healthy subjects: a is male; b is female.

Figure 6 is a correlation of CS levels in serum of sepsis patients with a SOFA score.

FIG. 7 is a graph of the blood serum CS content of patients with sepsis versus ROC curve analysis for healthy subjects and patients with septic shock; a is the result of comparing a sepsis patient with a healthy subject; and B is the result of comparing the patient with sepsis with the patient with sepsis shock.

FIG. 8 shows the expression of AST, ALT, TNF-. Alpha.in serum of CLP-operated mice: a is ALT; b is AST; c is TNF-alpha.

FIG. 9 shows the CS content in serum of CLP-treated mice.

Detailed Description

The examples provide experimental methods and results for supporting and validating the animal models used in the present invention. The relevant cases all used appropriate control experiments and statistical analysis methods. The following examples are intended to illustrate, but not limit, the application of the present invention. The methods and techniques involved in these cases can be used to determine the marker effect of cholesterol sulfate. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The study population is as follows:

blood of sepsis patients diagnosed by western university of Sichuan Huaxi hospital at 10-12 months 2021 was collected as a test group, blood of health examinees at the same period was collected as a healthy control group, and the test group had 16 (52%) and 15 (48%) cases for men and women, and 7 (54%) and 6 (46%) cases for men and women. Placing the blood sample in a 4 ℃ centrifuge, rotating at 3000rpm, centrifuging for 10 minutes, taking supernatant, freezing and storing at-80 ℃, and detecting the content of cholesterol sulfate in serum by liquid chromatography-tandem mass spectrometry. All patients met the diagnostic criteria Sepsis3.0 for the disease at the International conference on sepsis. SOFA scoring was performed according to FIG. 1. Subjects all studies were approved by the scientific and ethical committee of the western hospital, university of sichuan, sichuan.

Experimental animals:

c57BL/6J male mice at 8 weeks of age, weighing 18-22g, were purchased from Beijing Huafukang Biotech GmbH. All animals were housed in clean-grade animal houses. The experimental mice were divided into a sham group (sham group), a surgery group (CLP group). Performing Cecal Ligation and Perforation (CLP) operation on CLP group mice, and establishing a sepsis mouse short-term survival model: after anesthetizing the mouse with pentobarbital sodium, the abdominal cavity is opened, the cecum is exposed, the cecum is ligated from the tail end of the cecum to the ileocaecal region at a position of 0.75cm, the cecum is punctured twice with an 18-G needle after ligation to cause the leakage of the cecum content, and finally the cecum is inserted back into the abdominal cavity and sutured. Sham group mice were operated by opening the abdominal cavity and isolating the exposed cecum, but without the ligation and perforation procedure, the skin was sutured directly. After the operation, 1ml of physiological saline is injected subcutaneously as a fluid replacement, and the supporting treatment such as antibiotics is not used. Blood samples were collected from mice 24 hours post-surgery.

Example 1 study of relationship between CS content in serum and sepsis

1. Determination of CS content in serum of sepsis patient and serum of CLP mouse

1. LC-MS/MS conditions were as follows:

(1) Chromatographic conditions

The gradient elution mode is adopted, and the chromatographic conditions are as follows:

elution protocol:

(2) Conditions of Mass Spectrometry

Electrospray ionization source (ESI), negative ion scanning mode, GAS1:60psi, GAS2:60psi, curtain GAS:10psi, source temperature 600 ℃, ion spray voltage (IS) 4500V, declustering voltage (DP) 100V, ion pair of CS 465.1-96.9; the collision energy was 43eV, the ion pair for the internal standard was 265.1-223.1, and the collision energy was 45eV.

2. Sample pretreatment

Serum of clinical normal healthy people and sepsis patients is collected for analysis.

Blood was collected from mice in CLP and sham groups, serum was extracted by centrifugation in heparinized EP tubes at 3000rpm for 10 minutes at 4 ℃ and 30. Mu.L of the serum was transferred to 1.5mL EP tubes; 120 mu L of internal standard working solution (honokiol methanol solution) with the concentration of 200ng/mL is sucked by a retropipetting gun and added into a serum sample, the mixture is evenly mixed by vortex, the mixture is centrifuged for 15 minutes at 13000rpm, 80 mu L of supernatant is sucked and transferred into a disposable inner cannula in a sampling bottle, and 0.2 mu L of internal standard working solution is sucked by an automatic sample injector for quantitative analysis.

3. Establishment of a Standard Curve

Precisely sucking 3 mu L of CS reference substance solution with the concentration of 1, 0.3, 0.1, 0.03 and 0.01 mu g/mL into 5 EP tubes, respectively adding 3 mu L of deionized water and 120 mu L of internal standard precipitant (200 ng/mL of honokiol methanol solution), vortex and mixing uniformly, centrifuging at 13000rpm for 15 minutes, sucking 80 mu L of supernatant, transferring to a disposable inner intubation in an entry bottle, and sucking 0.2 mu L by an automatic sample injector. The standard curve equation (FIG. 2) was obtained by performing a linear regression operation using the weighted least square method (weight coefficient of 1/X2) with the concentration of CS as the abscissa X (. Mu.g/mL) and the area ratio of CS to IS peak (A1/A2) as the ordinate Y. The internal standard and standard peak times are shown in FIG. 3.

4. Analysis of results

And (3) collecting the chromatogram by using an Analyst 1.6.2 software workstation, integrating the object to be detected and the internal standard compound and recording the chromatographic peak area.

2. Determination of ALT, AST and TNF-alpha content in serum

The serum ALT, AST and TNF-alpha content of the mice was monitored by ELISA.

3. Statistical method

Results are all expressed as mean ± standard deviation. The comparison between the data was examined using unpaired t-test or one-way anova multiple comparison test or binary regression equation. When P is less than 0.05, the statistics difference exists.

4. Results of the experiment

1. Data statistics for patients with sepsis and physical examination persons

Data statistics for patients with sepsis and healthy subjects are shown in figure 4. From FIG. 4, it can be seen that all the subjects (sepsis patients) met the sepsis diagnosis standard, and the white blood cells and CRP were significantly increased.

2. Detection result of CS content in serum of sepsis patient

FIG. 5 is a graph showing the results of measuring the CS expression level in the serum of male and female sepsis patients. The results show that: compared with healthy subjects, the CS level is obviously increased in sepsis patients (p is less than 0.05), and the phenomenon is observed in both male and female patients, which indicates that the CS content in serum of the sepsis patients can be used as an index for early auxiliary diagnosis of sepsis; the patients with the Sepsis are divided into a non-shock group (Sepsis) and a shock group (Sepsis-shock), and the fact that the CS content is remarkably reduced after the Sepsis shock occurs along with the severe progress of the Sepsis is found. And (4) supposing according to the result: in the early stage of sepsis, CS expression is significantly increased in serum, but as sepsis progresses, CS is gradually consumed, its levels fall back, and the CS content in serum is significantly reduced. According to the results, the patients with sepsis clinically determined can be analyzed according to the CS content in the serum, namely the CS content in the serum can be used for auxiliary diagnosis, auxiliary treatment result monitoring and auxiliary prognosis evaluation of sepsis.

3. Correlation analysis of CS content in serum of sepsis patients and SOFA score

Figure 6 shows that CS levels in serum of 31 sepsis patients appear negatively correlated with the SOFA score. And, the higher the SOFA score, the more disease state, and the lower the level of CS expression. Thus, CS can be used as an indicator of clinical sepsis diagnosis and to indicate the severity of sepsis. Clinically, the SOFA score is a great index for judging the severity of the disease and the prognosis of the disease, and the higher the SOFA score is, the heavier the severity of the disease is, and the worse the prognosis is, so that the CS content in the serum of a patient can be used for carrying out auxiliary evaluation on the severity of the sepsis, the treatment effect and the prognosis of the patient.

CS is inversely correlated with SOFA levels, and normally, CS expression is constant, similar to blood triglycerides, but in septic patients CS expression is significantly higher than normal, but with disease severity and disease state changes CS may assume a state similar to decompensation. CS changes are observed in the same patient; only the changes in patients with sepsis are described here, and since it was subsequently observed that the sepsis lesions were greatly reduced when CS was supplemented in vivo, the effect of CS should be a protective effect at this point, increasing in the early stages of the disease, and decreasing significantly with changes in disease severity, possibly by exogenous replenishment.

4. ROC curve analysis of CS content in serum of sepsis patient, CS content in serum of healthy subject and CS content in serum of sepsis shock patient

As shown in fig. 7: the Area under the Curve (AUC) of ROC in FIG. 7A is 0.8846, and when the detection cut-off value (cut-off value) is 1.857, the specificity is 1 and the sensitivity is 0.55; the area under the curve (AUC) of ROC in FIG. 7B was 0.7773, and when the detection cut-off (cut-off) was 1.047, the specificity was 1 and the sensitivity was 0.3636. The results show that the specificity of the auxiliary diagnosis of the sepsis by using the CS content in the serum is good.

5. The serum content of AST, ALT and TNF-alpha of CLP operation mice is obviously increased

FIG. 8 shows the results of the measurement of AST, ALT, TNF-alpha expression in the serum of CLP-operated mice, showing that: compared with a control group (sham), the serum contents of AST, ALT and TNF-alpha of mice in the CLP operation group are obviously increased (p is less than 0.05). Prompting the successful molding of the sepsis mouse.

6. Significant increase of CS level in serum of CLP operation mouse

FIG. 9 shows the result of the measurement of the CS expression content in the serum of CLP-operated mice, showing that: compared with a control group (sham), the CS content in the serum of the mice in the CLP operation group is obviously increased (p is less than 0.05), and the phenomenon is consistent with the phenomenon observed in a clinical sample. The suggestion shows that the CS content in the serum can be used as an index for auxiliary diagnosis of sepsis.

Example 2 kit composition for detecting the content of CS in serum of patients with sepsis and method of use thereof

Based on the detection of the CS content in the serum of a sepsis patient, a kit for assisting in sepsis diagnosis, assisting in monitoring the sepsis treatment effect and assisting in sepsis prognosis evaluation can be developed. The detection is carried out by a liquid chromatography tandem mass spectrometry method (LC-MS/MS) by using the kit.

1. Composition of the kit of the invention

(1) An aqueous solution containing 0.01% ammonia; (2) acetonitrile; and (3) a magnolol standard substance.

2. Kit using method

According to the liquid chromatography tandem mass spectrometry method in the embodiment 1, the CS content in serum is detected by adopting the conditions of the chromatogram and the mass spectrometry in the embodiment 1, and sepsis auxiliary diagnosis is carried out on a sepsis patient, or the treatment effect of sepsis is monitored in an auxiliary way, or the prognosis of sepsis is evaluated in an auxiliary way:

for patients with sepsis, when the detected value of the CS content in serum is more than 1.857 mug/ml, determining that the patients have sepsis; when the detected value of the CS content in the serum is less than 1.047 mug/ml, the disease is judged to be developed into the septic shock state as the disease state of the patient with the sepsis changes.

The kit can realize the auxiliary diagnosis of the sepsis and the auxiliary monitoring of the development of the sepsis disease by quantitatively detecting the CS in the serum, can simply and quickly provide valuable reference information for clinicians, is convenient for symptomatic medication, and has good application prospect.

In conclusion, the research of the invention finds that the cholesterol sulfate can be used as an index for auxiliary diagnosis, auxiliary monitoring and auxiliary prognosis evaluation of sepsis: the content of cholesterol sulfate in early serum of patients with sepsis is obviously increased, and the patients enter a shock state along with aggravation of symptoms, so that the content of the cholesterol sulfate is reduced, and the cholesterol sulfate can be used as an auxiliary index for evaluating the severity of the sepsis. In addition, the treatment effect of the sepsis can be monitored in an auxiliary way and the prognosis evaluation can be carried out according to the expression level of cholesterol sulfate during the treatment process. The kit is used for auxiliary diagnosis, auxiliary monitoring of treatment effect and auxiliary prognosis evaluation of sepsis by detecting cholesterol sulfate in serum, provides a method for auxiliary monitoring of clinical sepsis, provides effective basis for patients to take relevant treatment measures or decisions, and has good clinical application prospect.

Claims

1. The application of the reagent for detecting the cholesterol sodium sulfate in preparing a kit for sepsis auxiliary diagnosis, treatment effect auxiliary monitoring and/or auxiliary prognosis evaluation;

the reagent for detecting the cholesterol sodium sulfate salt is a reagent for detecting the content of the cholesterol sodium sulfate salt in human blood;

the detection method of the reagent for detecting the cholesterol sodium sulfate salt adopts liquid chromatography tandem mass spectrometry detection;

the chromatographic detection conditions are as follows:

stationary phase Agilent Exten-C18 column Mobile phase A Aqueous solution containing 0.01% ammonia Mobile phase B Acetonitrile Column temperature 30℃ Flow rate of flow 0.5mL/min Sample volume 0.2μL Internal standard Honokiol standard substance

；

The elution mode of the chromatographic detection is gradient elution, and the elution conditions are as follows:

Time mobile phase A Mobile phase B 0.0 min 60% 40% 1.0 min 98% 2% 2.2 min 98% 2%

；

The mass spectrum detection conditions are as follows:

electrospray ionization source, negative ion scanning mode, GAS1:60psi, GAS2:60psi, curtain GAS:20psi, source temperature 500 ℃, ion spray voltage (IS) 4500V, declustering voltage (DP) 100V, ion pair of CS 465.1-96.9; the collision energy was 43eV, the ion pair for the internal standard was 265.1-223.1, and the collision energy was 45eV.

2. Use according to claim 1, characterized in that: the reagent for detecting the cholesterol sodium sulfate salt is a reagent for detecting the content of the cholesterol sodium sulfate salt in human serum.

3. A sepsis auxiliary diagnosis, treatment effect auxiliary monitoring and/or auxiliary prognosis evaluation kit is characterized in that: it comprises the reagent for detecting sodium cholesteryl sulfate according to claim 1 or 2.