Use of cholesteryl sulfate in the manufacture of an analytical reagent or kit for the diagnosis and course assessment of COVID-19

Abstract

The present invention relates to the use of cholesteryl sulfate in the manufacture of an analytical reagent or kit for the diagnosis or course assessment of COVID-19. The present invention also provides a method for the diagnosis or course assessment of COVID-19 for non-diagnostic and therapeutic purposes. The present invention provides a method for the diagnosis or course assessment of COVID-19 for non-diagnostic and therapeutic purposes, which is to detect the relative expression level of cholesteryl sulfate in serum as a biomarker by using the kit.

Description

Technical Field

[0001] This invention relates to the use of cholesterol sulfate in the preparation of analytical reagents or kits for the diagnosis and course assessment of COVID-19. Background Technology

[0002] Cholesterol sulfate (CS) is a major steroid sulfate in human blood plasma, formed by replacing the hydrogen atom at the third position of the cholesterol molecule with a sulfoxy group. CS is not only present in various bodily fluids such as blood, urine, semen, and bile, but it is also a normal component of different tissues including the skin, aorta, adrenal glands, liver, sperm, platelets, and red blood cells. Recent studies have found that CS can act as a signaling molecule, playing important roles in regulating cellular oxidative stress, glycolipid metabolism, inflammation, immunity, and survival.

[0003] Previous literature reports the use of cholesterol sulfate for the prevention of sepsis (Application No.: 202210040884.8, Invention Title: Use of Cholesterol Sulfate in the Preparation of Drugs for the Prevention of Sepsis). This invention provides the use of cholesterol sulfate in the preparation of drugs for the prevention of sepsis. Animal experiments demonstrate that sodium cholesterol sulfate can effectively prevent sepsis and its complications, protect the liver, kidneys, lungs, and heart, and significantly reduce the levels of TNF-α and IL-6, the main inflammatory factors, in the serum of septic mice, thus producing a significant protective effect on the body. Injection of this sodium cholesterol sulfate can significantly improve the survival rate and survival time of septic mice.

[0004] There are currently no reports of using CS as a diagnostic marker for diseases. Summary of the Invention

[0005] The technical solution of this invention provides a new use for cholesterol sulfate as a disease marker.

[0006] This invention provides the use of cholesterol sulfate in the preparation of analytical reagents or kits for the diagnosis and course assessment of COVID-19.

[0007] The cholesterol sulfate is 5-cholestene-3β-ol sulfate or its pharmaceutically acceptable salt, stereoisomer, or solvent compound; the structural formula of the sodium salt of 5-cholestene-3β-ol sulfate is:

[0008]

[0009] The cholesterol sulfate content was significantly elevated in the serum of COVID-19 patients.

[0010] The cholesterol sulfate content was higher in severe COVID-19 cases than in mild COVID-19 cases.

[0011] The present invention also provides a COVID-19 diagnostic or disease course assessment kit, which includes reagents for the specific detection of cholesterol sulfate.

[0012] This invention provides a method for diagnosing or assessing the course of COVID-19 for non-diagnostic and non-therapeutic purposes, which uses the kit described above to detect the relative expression level of cholesterol sulfate in serum as a biomarker. Attached Figure Description

[0013] Figure 1 Serum lactate (CS) analysis in COVID-19 patients and healthy volunteers (A. Serum CS levels in healthy volunteers, COVID-19 patients, and severely ill COVID-19 patients; B. Correlation between serum lactate levels and CS in severely ill COVID-19 patients; D. Correlation between oxygenation index and CS in severely ill COVID-19 patients; * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001);

[0014] Figure 2 Analysis of serum CS ROC curves in COVID-19 patients (A. ROC curves of healthy individuals and COVID-19 patients). Detailed Implementation

[0015] Example 1: Analysis of peripheral blood CS levels and COVID-19 severity according to the present invention

[0016] 1. Detection method: Peripheral blood CS levels were detected by mass spectrometry combined with chromatography.

[0017] The conditions for mass spectrometry-chromatographic detection of CS are as follows:

[0018] The chromatographic conditions for detecting CS 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: 0.5 mL / min; injection volume: 0.2 μL; elution conditions are as follows:

[0019]

[0020]

[0021] Mass spectrometry detection conditions: electrospray ionization source, negative ion scanning mode, GAS1: 60 psi, GAS2: 60 psi, Curtain GAS: 20 psi, source temperature: 501℃, ion spray voltage (IS): 4550 V, declustering voltage (DP): 105 V, CS ion pair: 465.1-96.9; collision energy: 42 eV, internal standard ion pair: 265.1-223.1, collision energy: 46 eV.

[0022] Data processing and statistical analysis

[0023] Experiments were repeated at least three times to obtain similar results. Results are expressed as mean ± standard deviation. Data comparisons were performed using unpaired t-tests or one-way ANOVA multiple comparison tests. A p-value < 0.05 was considered statistically significant.

[0024] 2. Collection of blood samples from clinical COVID-19 patients

[0025] Inclusion criteria: COVID-19 patients with obvious signs of viral infection in nucleic acid testing, antigen testing, or chest CT scan. Ultimately, 12 healthy volunteers and 44 COVID-19 patients (including 26 (59%) COVID-19 patients with no other symptoms and 18 (41%) COVID-19 patients with severe symptoms) were included.

[0026] 3. Analysis of serum CS levels in COVID-19 patients

[0027] Serum lactate is an important indicator reflecting tissue hypoxia and hypoperfusion in critically ill patients. The oxygenation index (OI), the ratio of arterial partial pressure of oxygen to inhaled oxygen concentration, has a normal range of 400-500 mmHg and is an important target for respiratory therapy. A value below 200 mmHg can diagnose acute respiratory distress syndrome (ARDS) and is a crucial indicator for clinically assessing the prognosis of severe pneumonia. Serum lactate (CS) levels were detected using mass spectrometry combined with chromatography. Compared with healthy volunteers, serum CS levels in COVID-19 patients were significantly elevated. Figure 1 A)(P<0.001); Compared with patients with simple COVID-19, serum CS levels were also significantly increased in patients with severe COVID-19 (P<0.01). Analysis of serum lactate in patients with severe COVID-19 revealed a positive correlation between CS levels and lactate levels. Figure 1 B)(P=0.0156, r=0.6108); Analysis of clinical data of severely ill COVID-19 patients and calculation of oxygenation index revealed that as oxygenation index increased, CS content decreased, and the two showed a negative correlation. Figure 1 C)(P=0.0182, r=-0.5992).

[0028] 4. ROC curve analysis of blood CS levels in COVID-19 patients and healthy subjects

[0029] Figure 2 As shown, the area under the ROC curve (AUC) is 0.8269; when the detection cut-off value is 538.5, its specificity is 0.4167 and its sensitivity is 1. This suggests that CS has high diagnostic value for both healthy individuals and COVID-19.

[0030] Analysis of peripheral blood samples from COVID-19 patients revealed a significant increase in serum lactate (CS) levels. CS levels further increased as COVID-19 patients progressed to severe illness. Further analysis of serum lactate and oxygenation index showed that CS levels increased with rising lactate levels and decreased with rising oxygenation index. CS levels were positively correlated with the severity of COVID-19 illness.

Claims

1. Use of cholesterol sulfate in the preparation of analytical reagents or kits for the diagnosis or course assessment of COVID-19; wherein the cholesterol sulfate is 5-cholestyrene-3β-ol sulfate or a pharmaceutically acceptable salt, stereoisomer, or solvent compound thereof; the structural formula of the sodium salt of 5-cholestyrene-3β-ol sulfate is:

2. The use according to claim 1, characterized in that: The cholesterol sulfate content mentioned above was significantly elevated in the serum of COVID-19 patients.

3. The use according to claim 2, characterized in that: The cholesterol sulfate content was higher in severe COVID-19 cases than in mild COVID-19 cases.

4. A COVID-19 diagnostic or disease course assessment kit, characterized in that: It includes reagents for the specific detection of cholesterol sulfate; The cholesterol sulfate is 5-cholestene-3β-ol sulfate or its pharmaceutically acceptable salt, stereoisomer, or solvent compound; the structural formula of the sodium salt of 5-cholestene-3β-ol sulfate is:

5. A method for diagnosing or assessing the course of COVID-19 for non-diagnostic and non-therapeutic purposes, characterized in that: It uses the kit described in claim 4 to detect the relative expression level of cholesterol sulfate in serum as a biomarker.

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