Application of LPC, PC and AA in children sepsis diagnosis and prognosis product development

Abstract

The invention relates to the field of biological medicine, and discloses application of LPC, PC and AA in children sepsis diagnosis and prognosis product development. According to the invention, a high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) technology is used, plasma samples of a child sepsis patient and a healthy control group are compared, the relationship between lipidome change and pSOFA score is analyzed, and specific lipid molecules closely related to disease severity and prognosis are identified. According to the invention, the following lipid molecules are highlighted as key biomarkers: lysophosphatidylcholine (LPC), phosphatidylcholine (PC) and arachidonic acid (AA). The change of the lipid molecules is closely related to the physiological state of a child sepsis patient, and the lipid molecules can be used for early diagnosis and evaluation of the state of the child sepsis, so that the treatment effect and the accuracy of prognosis evaluation are improved, and a new treatment strategy and a new management method are provided for clinic.

Description

Technical Field

[0001] The present invention relates to the application of specific lipid molecules LPC, PC and AA in the development of products for the diagnosis and prognosis of pediatric sepsis, and belongs to the field of biomedical technology. Background Art

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Despite significant advances in healthcare that have led to improved clinical outcomes and a steady decline in mortality, severe sepsis remains the leading cause of death in pediatric intensive care units (PICUs). Alarmingly, refractory shock accounts for one-third of early deaths. Despite some progress in antibiotic treatment and infection control, the early identification and effective treatment of sepsis remain clinical challenges. Many children who survive sepsis may develop physical, cognitive, emotional, and other disorders, severely affecting their quality of life and imposing a huge burden on many families.

[0003] New metabolomics studies have revealed a key role of lipid metabolism in the pathophysiology of sepsis. Sepsis induces severe lipid metabolic disorders, characterized by elevated levels of certain lipids such as triglycerides (TG) and phosphatidylserine (PS), and these changes can in turn lead to dysregulated inflammatory responses. These lipids are not only markers of metabolic imbalance but also key signaling molecules that regulate cell recognition, immune responses, and overall disease progression, having a significant impact on the prognosis of patients. Most studies have focused on changes in lipoprotein composition and content, cholesterol distribution, and the diagnostic potential of lipidomic biomarkers. During sepsis, the interaction between lipopolysaccharide (LPS) and lipoproteins enhances the lipoprotein-mediated clearance mechanism while activating the activity of scavenger receptors. This results in a rapid decline in lipoprotein levels. This process also leads to a decrease in cholesterol levels, hypertriglyceridemia, and an increase in fatty acid content. Therefore, understanding lipid metabolism may provide a new perspective for revealing disease mechanisms, prognosis, and potential therapeutic targets.

[0004] Untargeted lipidomics is a method for comprehensively detecting lipids in plasma by high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) technology. This method can widely analyze various lipid molecules present in plasma samples, including different types of fatty acids, phospholipids, etc., thus providing a powerful tool for disease diagnosis and biomarker discovery. Pediatric sequential organ failure assessment (pSOFA) has a strong correlation with in-hospital mortality and is a valuable tool for assessing the disease severity of pediatric sepsis patients.

[0005] Phosphatidylcholine (PC) can act as a potential inflammatory mediator by disrupting cell membrane stability and exacerbating the inflammatory response in patients. Abnormally high or low PC / PE ratios in cells can affect the energy metabolism of various organelles, and these changes are related to the disease progression of many tissues. A decrease in the liver PC / PE ratio impairs membrane integrity and leads to the development of NAFLD and even liver failure. In the intestine, it can inhibit the uptake of intestinal fatty acids and the secretion of chylomicrons. The PC / PE in skeletal muscle can affect the activity of SERCA, disrupt cellular calcium homeostasis to affect muscle insulin sensitivity, and inhibit muscle contraction. Lysophosphatidylcholine (LPC) is a product of PC degradation mediated by phospholipase A2 (PLA2) and is closely related to inflammation. As a bioactive lipid, LPC can not only act as a ligand for lymphocytes but also be enzymatically converted into lysophosphatidic acid, which is an anti-inflammatory mediator. Persistently low LPC levels in septic patients are thought to reflect a disruption of metabolic homeostasis. These lower LPC levels are associated with excessive inflammatory responses and poor clinical outcomes, highlighting the key role of LPC in the pathophysiology of sepsis. Arachidonic acid (AA), as an important precursor of inflammatory mediators, plays a central role in the systemic inflammatory response (SIRS) triggered by sepsis. During the acute phase of sepsis, the accelerated metabolism of AA leads to the production of pro-inflammatory mediators such as prostaglandins and leukotrienes, thereby enhancing the immune response. Elevated AA levels have been observed in patients with severe inflammatory responses, highlighting its potential as a biomarker of disease activity.

[0006] Although significant progress has been made in understanding lipid metabolism in adult sepsis, research on pediatric sepsis remains scarce. Many previous studies have been based on changes in blood lipid profiles in adult patients with sepsis, and there is currently no systematic study on lipids in pediatric sepsis. Most previous studies have not fully elucidated the relationship between lipid metabolism disorders and clinical applications, and most of them have focused on specific lipid types, with insufficient analysis of the entire lipidome, limiting our comprehensive and in-depth understanding of the comprehensive role of lipids in sepsis. Summary of the Invention

[0007] The object of the present invention is to provide a novel strategy for the diagnosis and treatment of pediatric sepsis - the application of LPC, PC, and AA in the development of products for the diagnosis and prognosis of pediatric sepsis. Through non-targeted lipidomics analysis technology (UHPLC-MS / MS), the present invention identifies specific lipid molecules closely related to the severity and prognosis of sepsis, providing a new method and technical support for the accurate diagnosis and condition assessment of pediatric sepsis.

[0008] To achieve the above object, the present invention provides the use of LPC, PC, and AA in the preparation of products for the diagnosis or prognosis of pediatric sepsis; this method includes analyzing the association between lipids and the pSOFA score to evaluate the role of lipids in disease severity. By comparing the expression differences of specific lipids in sepsis patients with different prognoses, the function of lipids in the disease process is explored.

[0009] Specifically, the following technical solutions are adopted:

[0010] In a first aspect, the present invention provides the use of specific lipid molecules in the preparation of products for the diagnosis or prognosis of pediatric sepsis.

[0011] Furthermore, the specific lipid molecules include lysophosphatidylcholine (LPC), phosphatidylcholine (PC), and arachidonic acid (AA), and the changes in the levels of these molecules in the plasma of pediatric sepsis patients are significantly different from those in the control group.

[0012] Preferably, the correlation analysis of the changes in the identified specific lipid molecules with the pSOFA score further verifies their potential clinical value as biomarkers.

[0013] Preferably, the identified specific lipid molecules can be applied to the development of a diagnostic kit or a prognosis assessment tool for pediatric sepsis, which can provide a reliable reference basis for disease severity, treatment response, and disease prognosis.

[0014] In a second aspect, the present invention provides the use of LPC, PC, and AA in the preparation of products for the diagnosis or prognosis of pediatric sepsis.

[0015] Furthermore, the diagnostic or prognostic product includes a kit, and the kit can provide information about disease severity, treatment response, or disease prognosis.

[0016] Furthermore, the application of LPC, PC, and AA includes assisting in the diagnosis of pediatric sepsis and improving the prognosis of sepsis.

[0017] In a third aspect, the present invention provides a diagnostic or prognostic kit for pediatric sepsis, including reagents for detecting the levels of LPC, PC, and AA in patients.

[0018] Furthermore, the test sample is a plasma sample.

[0019] Among the specific lipid molecules of the present invention, the levels of LPC and PC show a strong correlation with the pSOFA score and are significantly reduced in sepsis patients with poor prognosis.

[0020] AA is significantly reduced in patients with poor prognosis of sepsis and shows a significant negative correlation with cholesterol.

[0021] In this invention, 4,144 lipid molecules of 44 lipid classes in the plasma of 48 children with PICU sepsis and 48 outpatient children without infection as the control group were analyzed. The results showed that the plasma lipidome of children with sepsis changed significantly, and 24 lipid molecules had a strong correlation with pSOFA. By analyzing the improved and deteriorated groups of sepsis, a total of 186 differential lipid molecules were screened out. The differences in lipid molecules related to pSOFA between the improved and deteriorated groups of sepsis were compared, and 15 overlapping lipid molecules were found, among which LPC and PC showed significant differences. In addition, it was found that the fatty acid profile of sepsis patients showed a significant change in arachidonic acid (FA(20:4)), and related diagnostic markers were screened out.

[0022] Different from past studies, this invention systematically studied the dynamic changes of lipid molecules in childhood sepsis for the first time, determined their relationship with disease severity, explored their changes in disease prognosis, and clarified the biomarker role of lipid molecules. This invention first discovered the strong correlation between LPC, PC, and AA and pSOFA and their significant changes in prognosis. Finally, it was determined that the development of diagnostic and prognostic products based on LPC, PC, and AA is a promising new method for diagnosing and treating sepsis.

[0023] Compared with the prior art, this invention has the following beneficial effects:

[0024] This invention first proposed the value of LPC, PC, and AA as diagnostic and prognostic markers for childhood sepsis, and verified the specificity and sensitivity of these lipid molecules through non-targeted lipidomics analysis technology. We found significant changes in blood lipids in sepsis patients, and these changes reflected disease severity, emphasizing the dynamic role of lipids in the host response to sepsis; among the identified lipid molecules, 15 were related to disease severity and clinical outcomes, and LPC and PC showed significant differences. In addition, this invention first proposed that fatty acid levels, especially arachidonic acid (FA(20:4)), showed a significant increase in poor sepsis prognosis, showing strong prognostic potential and highlighting their relevance as diagnostic markers; therefore, the application of lipid molecule markers provides new ideas for the treatment strategy of sepsis and promotes the development of precision medicine for childhood sepsis. Based on the lipid molecule (LPC, PC, and AA) diagnostic tool of this invention, it can quickly provide accurate information about disease severity and treatment response, improve the efficiency of clinical diagnosis and treatment, and has broad clinical application prospects and industrialization value. Description of the Drawings

[0025] Figure 1. Plasma lipidome changes are closely related to disease severity: (A) Pie chart of differentially screened lipids according to the results of PLS-DA; (B) Bar chart of lipids related to the pSOFA score; (C) Scatter plot of lipids related to the pSOFA score; (D) Correlation heat map of lipids related to the pSOFA score;

[0026] Figure 2 . Changes in the plasma lipidome of sepsis patients are associated with poor prognosis: (A) Box plot of all lipid classes in the improved and deteriorated sepsis groups; (B) Pie chart of differentially lipids in the improved and deteriorated sepsis groups; (C) Unsupervised hierarchical clustering heat map of differentially lipids in the improved and deteriorated sepsis groups; (D, E) Overlapping lipid molecules between differentially lipids and pSOFA-related lipids in the improved and deteriorated sepsis groups; (F) Bar chart of laboratory indicators in the improved and deteriorated sepsis groups.

[0027] Figure 3 . The plasma fatty acid profile of sepsis patients changes: (A) Stacked bar chart of fatty acids comparing the control group and the sepsis group; (B) Box plot of fatty acids in the control group and the sepsis group; (C) Box plot of fatty acids in the improved and deteriorated groups; (D) Correlation heat map of fatty acids and cholesterol esters in sepsis patients; (E) Scatter plot of the correlation between fatty acids and cholesterol esters;

[0028] Statistical significance was considered at p < 0.05; in the above figures, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Detailed implementation mode

[0029] To make the present invention more obvious and understandable, preferred embodiments are hereby described in detail in conjunction with the accompanying drawings as follows.

[0030] In the following examples, the experimental methods used are conventional methods unless otherwise specified; the experimental population used was approved by the Ethics Committee of Children's Hospital of Fudan University (

[2024] No. 116), and informed consent was obtained from the parents or guardians of all participants; the materials, reagents, etc. used are conventional commercially available products unless otherwise specified.

[0031] In the following examples, the experimental population and methods include:

[0032] 1. Research population

[0033] A total of 51 pediatric patients were recruited from the PICU of Fudan University Children's Hospital between June 2022 and May 2024. Inclusion criteria were PICU inpatients aged 28 days to 18 years within 24 hours of admission or 24 hours after new sepsis. Exclusion criteria included known lipid metabolism disorders, hyperlipidemia, parenteral nutrition, and severe liver dysfunction. According to the results within 28 days after the diagnosis of sepsis, the patients were divided into improvement group and deterioration group. Outpatient subjects without infection or acute illness and matched in gender and age during the same period served as the control group.

[0034] 2. Collection and Processing of Biological Samples

[0035] Blood samples (2 ml) were collected from all participants in EDTA anticoagulation tubes to obtain plasma. The samples were subjected to a two-step centrifugation process: the first centrifugation was at 400 × g for 10 min at 4 °C to remove cells, and then the second centrifugation was at 3000 × g for 10 min at 4 °C to obtain platelet-free plasma. The supernatant was divided into 200 μL portions, placed on dry ice, and stored at -80 °C for further analysis.

[0036] 3. Lipidomics Analysis

[0037] Non-targeted lipidomics analysis was performed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). Lipid sample analysis and relative quantification were performed by Shanghai Zhongke New Life Biotechnology Co., Ltd. (Relative Quantitative Lipidomics).

[0038] 4. Analysis of Differential Lipids

[0039] To identify differential lipid molecules in pediatric sepsis patients, we used screening criteria based on partial least squares discriminant analysis (PLS-DA) results. Lipids were considered significant if they met the following thresholds: projected variable importance (VIP) > 1, P < 0.05, and fold change (FC) > 1.5 or FC < 0.67.

[0040] 5. Statistical Analysis

[0041] All statistical analyses were performed using IBM SPSS Statistics for Windows, Version 25.0 (IBM Corp., Armonk, NY, USA). The t-test was used to compare continuous variables with a normal distribution, and non-parametric tests (Mann-Whitney U test) were used to analyze data with a non-normal distribution. The Spearman rank correlation test was used to examine the correlation between variables, and scatter plots were created using GraphPad Prism version 10.1 (GraphPad Software, San Diego, CA, USA) for visualization. To explore patterns in the lipidomics data, unsupervised hierarchical clustering and additional data visualization were performed using TBtools software (vLa-Π). The standard Benjamini-Hochberg method was used to control the false discovery rate (FDR) for multiple hypothesis testing correction. All statistical tests were two-sided, and P < 0.05 was considered statistically significant.

[0042] Experimental Example 1: Analyzing the changes in plasma lipidome and disease severity

[0043] A total of 1,257 different lipid molecules were identified ( Figure 1 A). Among them, 24 lipid molecules were significantly correlated with the pSOFA score ( Figure 1 B). LPC(16:1e), LPC(19:1), and LPC(15:0) ( Figure 1 C) were negatively correlated with the pSOFA score. Correlation analysis of the 24 differential lipid molecules found that Cer was negatively correlated with TG, LPC, CL, and GM3 ( Figure 1 D).

[0044] The above results revealed that the changes in plasma lipidome of sepsis patients were closely related to disease severity. The combined detection of LPC and the pSOFA score may help to identify sepsis earlier, evaluate its severity, and provide strong support for personalized treatment.

[0045] Experimental Example 2: Analyzing the association between changes in plasma lipidome of sepsis patients and prognosis

[0046] The lipid classes between the improved and deteriorated groups of sepsis patients were compared, and significant differences were found in FA and lysophosphatidylinositol (LPI), with both lipids increasing in the deteriorated group ( Figure 2 A). In the analysis of lipid subclasses, 186 differential lipids were identified between the two groups ( Figure 2 B). Unsupervised hierarchical clustering analysis showed that LPC(15:0), LPC(20:1), and LPC(19:1) were elevated in the improved group ( Figure 2C). Fifteen overlapping lipid molecules were found among the 24 lipid molecules related to the pSOFA score and the 186 differential lipid molecules between the two groups. The top four most important lipids were LPC(15:0), LPC(19:1), LPC(20:1), and PC(18:3e)( Figure 2 D, E). In addition, compared with the improved group, the platelet count of patients in the deteriorated group was significantly decreased, and the levels of total bilirubin, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were significantly increased( Figure 2 F).

[0047] The above results revealed that the changes in the plasma lipidome of sepsis patients were associated with a higher risk of death. LPC and PC might reveal key biomarkers in sepsis, helping with diagnosis, predicting prognosis, and providing clues for potential therapeutic targets.

[0048] Experimental Example 3 Analyzing the Changes in the Fatty Acid Profile of Sepsis Patients

[0049] Fatty acid profile analysis showed that compared with the control group, FA(16:0), FA(20:4), and FA(22:6) were elevated in sepsis patients and showed higher levels in the deteriorated sepsis group( Figure 3 A, B, C). In addition, correlation analysis between FA and cholesterol ester (ChE) showed different relationships, and FA(20:4) was negatively correlated with ChE(19:1)( Figure 3 D, E).

[0050] The above results revealed that AA showed higher levels in patients with poor prognosis of sepsis and there was a close connection between it and cholesterol levels. The relationship between the two played an important role in the immune response and inflammatory response of sepsis.

[0051] The above results indicated that LPC, PC, and AA, as key biomarkers, were closely related to disease severity and prognosis. These lipid molecules played a key role in the pathological process of sepsis and became new markers for diagnosing pediatric sepsis, predicting prognosis, and guiding treatment.

[0052] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the premise of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. Application of specific lipid molecules in the preparation of diagnostic or prognostic products for pediatric sepsis.

2. The use according to claim 1, characterized in that The specific lipid molecules include lysophosphatidylcholine (LPC), phosphatidylcholine (PC) and arachidonic acid (AA).

3. Application of LPC, PC and AA in the preparation of diagnostic or prognostic products for pediatric sepsis.

4. The use according to claim 3, characterized in that The diagnostic or prognostic products include kits that are capable of providing information regarding disease severity, treatment response, or disease prognosis.

5. The use according to claim 3 or 4, characterized in that: The applications of LPC, PC, and AA include assisting in the diagnosis of sepsis in children and improving the prognosis of sepsis.

6. A diagnostic or prognostic kit for sepsis in children, characterized in that: Includes reagents for measuring LPC, PC and AA levels in patients.

7. The kit according to claim 6, characterized in that The test sample is a plasma sample.

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