CN112147329A

CN112147329A - Use of ATP citrate lyase as a diagnostic marker for sepsis

Info

Publication number: CN112147329A
Application number: CN202010914154.7A
Authority: CN
Inventors: 王春霞; 张育才
Original assignee: SHANGHAI CHILDREN'S HOSPITAL
Current assignee: SHANGHAI CHILDREN'S HOSPITAL
Priority date: 2020-09-03
Filing date: 2020-09-03
Publication date: 2020-12-29
Anticipated expiration: 2040-09-03
Also published as: CN112147329B

Abstract

The invention relates to the field of diagnostic medicine, in particular to application of ATP citrate lyase as a sepsis diagnostic marker. The invention combines the mouse serum of a mouse endotoxemia model induced by Lipopolysaccharide (LPS) and the serum of a clinical child sepsis patient, finds free ACLY in the serum for the first time through the proteomic identification of the serum by mass spectrometry, and further confirms the clinical value of ACLY in identifying sepsis and evaluating sepsis prognosis in a biological sample of the patient.

Description

Use of ATP citrate lyase as a diagnostic marker for sepsis

Technical Field

The invention relates to the field of diagnostic medicine, in particular to application of ATP citrate lyase as a sepsis diagnostic marker.

Background

Sepsis is a major cause of patient death in intensive care units. Children with sepsis death account for 40% of all deaths worldwide in children under five years of age. Sepsis is defined as a dysfunction of a life-threatening organ caused by a dysregulated host immune response resulting from an infection. The immunopathogenesis of sepsis is abnormally complex, involving early inflammatory factor storm and late immunosuppression. Lymphopenia due to lymphocyte apoptosis is a feature of sepsis and is also a major cause of septic late immunosuppression.

In recent years, immune metabolism regulation becomes a research hotspot of immune response regulation, and brings new hopes for recogniting immunopathological mechanisms and immune regulation treatment methods of sepsis. Immune cell function is closely related to metabolism in immune cells, and molecules involved in immune metabolism during sepsis are likely to provide help for monitoring immune status and even predicting sepsis outcome.

How to quickly and accurately distinguish the immune state of the organism of a sepsis patient at an early stage is lack of an effective biomarker at present.

Disclosure of Invention

The invention relates to application of a quantitative detection agent of ATP citrate lyase in preparation of a kit for diagnosis, auxiliary diagnosis or prognostic analysis of sepsis.

Optionally, the quantitative detection reagent is used to perform any one of the following methods:

biological mass spectrometry, electrophoresis, chromatography, enzyme-linked immunosorbent assay, and dot blotting.

Optionally, the quantitative detection agent is an antibody specific for ATP citrate lyase.

Optionally, the specific antibody is a monoclonal antibody or a polyclonal antibody.

Optionally, the kit further comprises a sample treatment reagent, wherein the sample treatment reagent comprises at least one of a sample lysis reagent, a sample purification reagent and a sample nucleic acid extraction reagent.

Optionally, the sample is selected from at least one of blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate, semen, and saliva samples of the subject.

Optionally, the kit further comprises at least one additional reagent for use in a diagnostic, auxiliary diagnostic or prognostic assay for sepsis.

Optionally, the reagent is selected from the group consisting of for detecting at least one of:

lactic acid, C-reactive protein, interleukin-6, tumor necrosis factor-alpha, PCT, sICAM-1, supAR, plasma circulating DNA, bacterial DNA, and miRNA.

Optionally, the reagent is used for blood culture or blood routine detection.

Optionally, the sepsis is childhood sepsis.

The invention has the beneficial effects that:

the invention combines the mouse serum of a mouse endotoxemia model induced by Lipopolysaccharide (LPS) and the serum of a clinical child sepsis patient, finds free ACLY in the serum for the first time through the proteomic identification of the serum by mass spectrometry, and further confirms the clinical value of ACLY in identifying sepsis and evaluating sepsis prognosis in a biological sample of the patient.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the results of mass spectrometric analysis of mouse serum proteomics in one embodiment of the present invention;

FIG. 2 is a graph showing that LPS induces an increase in serum ACLY levels in mice in one embodiment of the present invention;

FIG. 3 is a comparison of serum ACLY levels in healthy children and sepsis patients in one embodiment of the present invention;

FIG. 4 is a graph of the efficacy of a ROC curve analysis of serum ACLY diagnosis in identifying sepsis in one embodiment of the present invention;

FIG. 5 is a graph of serum ACLY levels when survivors and deaths of sepsis patients enter the PICU in one embodiment of the present invention;

FIG. 6 is a graph of the efficacy of ROC curve analysis of serum ACLY to assess sepsis prognosis in one embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.

The invention provides a novel marker for sepsis diagnosis and prognosis evaluation: ATP-citrate lyase. ATP-citrate lyase (English, ATP citrate lyase, citric acid lyase for short) is an enzyme which catalyzes an important step in fatty acid biosynthesis. This step occurs in fatty acid synthesis because ATP-citrate lyase is a bridge between sugar metabolism (energy production) and fatty acid production. ATP-citrate lyase is the major enzyme responsible for the production of acetyl-CoA in the cytosol in many tissues. The enzyme is a tetramer consisting of four apparently equal subunits. Although the role of ATP-citrate lyase is important, the main role is to participate in fatty acid biosynthesis, and the correlation between the ATP-citrate lyase and sepsis is not found in the prior art.

Animal experiments verify that free ACLY in serum is found for the first time through serum proteomic identification by mass spectrometry of mouse serum of a mouse endotoxemia model induced by LPS and clinical children sepsis patient serum, and the clinical value of ACLY in identification of sepsis and evaluation of sepsis prognosis is further confirmed in a patient biological sample.

The term "marker" as used herein refers to a molecule to be used as a target for the analysis of a patient test sample. Examples of such molecular targets are proteins or polypeptides. Proteins or polypeptides for use as markers in the present invention are intended to include naturally occurring variants of said proteins as well as fragments, in particular immunologically detectable fragments, of said proteins or of said variants. The immunologically detectable fragment preferably comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 15 or 20 consecutive amino acids of the marker polypeptide. One skilled in the art will recognize that proteins released by cells or present in the extracellular matrix may be damaged (e.g., during inflammation) and may be degraded or cleaved into such fragments. Certain markers are synthesized in an inactive form, which can be subsequently activated by proteolysis. As will be appreciated by the skilled artisan, proteins or fragments thereof may also be present as part of a complex. Such complexes may also be used as markers in the sense of the present invention. In addition, or in the alternative, the marker polypeptide or variant thereof may carry post-translational modifications. Non-limiting examples of post-translational modifications are glycosylation, acylation and/or phosphorylation.

In some embodiments, the quantitative detection reagent is used to perform any one of the following methods:

the method comprises the following steps of biological mass spectrometry, electrophoresis, chromatography, enzyme-linked immunosorbent assay, immunofluorescence, immunochemiluminescence, immunoturbidimetry, immunoblotting and dot blotting.

The quantitative detection agent is typically a reagent that specifically detects an ATP-citrate lyase, e.g., a ligand or receptor for ATP-citrate lyase, if present, a lectin that binds to ATP-citrate lyase, an aptamer that binds to ATP-citrate lyase, or an antibody and antibody fragment that binds to ATP-citrate lyase. The specific binding agent has at least 10 for its corresponding target molecule⁷Affinity of l/mol. The specific binding agent preferably has 10 to its target molecule⁸l/mol, or more preferably 10⁹Affinity of l/mol. The skilled person will understand that the term "specific" is used to indicate that other biomolecules present in the sample do not bind significantly to the quantitative detector of ATP-citrate lyase.

In some embodiments, the quantitative detection agent is an antibody specific for ATP citrate lyase.

In some embodiments, the specific antibody is a monoclonal antibody or a polyclonal antibody.

In some embodiments, the kit further comprises sample treatment reagents comprising at least one of sample lysis reagents, sample purification reagents, and sample nucleic acid extraction reagents.

In some embodiments, the sample is selected from at least one of blood, serum, cerebrospinal fluid, tissue or tissue lysate, semen, and saliva samples of the subject.

As used herein, "tissue lysate" may also be used in common with the terms "lysate", "lysed sample", "tissue or cell extract", and the like, to denote a sample and/or biological sample material comprising lysed tissue or cells, i.e., where the structural integrity of the tissue or cells has been disrupted. To release the contents of a cell or tissue sample, the material is typically treated with enzymes and/or chemical agents to lyse, degrade, or disrupt the cell walls and membranes of such tissues or cells. The skilled artisan is well familiar with suitable methods for obtaining a lysate. This process is encompassed by the term "lysis".

In some embodiments, the kit further comprises at least one additional reagent for use in a diagnostic, auxiliary diagnostic, or prognostic assay for sepsis.

In some embodiments, the reagent is selected from the group consisting of for detecting at least one of:

lactic acid, C-reactive protein (CRP), interleukin-6, tumor necrosis factor-alpha, Procalcitonin (PCT), sICAM-1, supAR, plasma circulating DNA, bacterial DNA, and miRNA.

In some embodiments, the reagents are used to perform blood culture or blood routine tests.

An ideal scenario for diagnosis is a situation where a single event or process may cause various diseases, e.g. in infectious diseases. In all other cases, correct diagnosis can be very difficult, especially when the etiology of the disease is not fully understood, as in the case of many cancer types. As the skilled artisan will appreciate, diagnosis without biochemical markers is 100% specific and with the same 100% sensitivity for a given multifactorial disease. Conversely, biochemical markers or indicators (e.g., lactate, CRP, interleukin-6, tumor necrosis factor-alpha, PCT, sICAM-1, suPAR, plasma circulating DNA, bacterial DNA, and miRNA or ATP-citrate lyase as demonstrated herein, blood culture or blood routine) can be used to assess the presence or severity of, for example, sepsis with some likelihood or predictive value. Thus, in routine clinical diagnosis, a combination of various clinical symptoms and biological markers is often considered to diagnose, treat and control underlying diseases.

Diagnosis of sepsis may include, but is not limited to, the following symptoms; tachypnea, tachycardia, hypotension, hyperthermia, or hypothermia, altered mental status, metabolic acidosis, impairment of kidney and liver function (as determined by an increase in white blood and urinary nitrogen) and ultimately leading to loss of muscle mass. Blood cultures were used to determine the presence of infectious microorganisms. Infectious microorganisms known to cause sepsis include, but are not limited to: gram-negative bacteria such as escherichia coli (e.coli), klebsiella, pseudomonas aeruginosa, and enterobacter; and gram-positive bacteria such as Staphylococcus epidermidis and Streptococcus faecalis. The diagnostic methods disclosed herein can be used in conjunction with the diagnostic methods described above.

In some embodiments, the sepsis is childhood sepsis.

According to a further aspect of the invention there is also provided a method for the diagnosis, co-diagnosis or prognostic assay of sepsis, the method comprising: the amount of ATP citrate lyase is measured using a quantitative detector as described above.

Embodiments of the present invention will be described in detail with reference to examples.

Examples

The first test method comprises the following steps:

1. preparing experimental animals and models:

male 8-week-old C57BL/6J mice, 30, were purchased from Shanghai laboratory animals, Inc. (Shanghai, China). Mice were kept in standard housing in clean animal houses and a mouse endotoxemia model was constructed with 10mg/kg LPS (E.coli 0111: B4). 30 male C57BL/6J mice were randomly divided into 3 groups (10 per group) including control group, LPS-treated 2h group (LPS 2h), LPS-treated 24 group (LPS 24 h). Mice were either treated with 0.9% physiological saline as a control, or were injected intraperitoneally with LPS (10 mg/kg). Mice were anesthetized with pentobarbital (0.2mg/kg) intraperitoneally at 2 or 24 hours post-LPS administration. Blood was collected from the heart, centrifuged at 3000rpm for 10 minutes, and serum was separated. Serum samples were stored at-80 ℃ for further analysis.

2. Serum proteomics analysis:

to improve the accuracy and precision of data in proteomic studies, we mixed equal amounts of 3 different samples into one pool for three pools (control, LSP 2h, LPS 24h, each group n 3). Serum proteomics analysis was performed with the aid of the digital spectrum biotechnology limited.

3. Serum sample of childhood sepsis:

sepsis patients who visited the PICU in this hospital in 2018 were screened in this study. The diagnosis of childhood sepsis is based on the 2005 international conference on pediatric sepsis consensus. Inclusion criteria 1) age 1 month to 18 years, 2) diagnosis of sepsis at admission, 3) PICU hospitalized for more than 24 hours. Exclusion criteria included 1) advanced tumors or life expectancy of less than 1 month, 2) congenital heart disease, 3) severe primary disease or inherited metabolic disease. As a control group, we analyzed 30 blood samples from physical examinations of healthy children, which were all normal in blood count, C-reactive protein (CRP), and liver enzymes. The study protocol was approved by the local ethical committee and was carried out according to the ethical standards as specified in the declaration of helsinki (approval No.: 2018R 039-F01). The informed consent was signed by the patient's parent or relative.

4. Enzyme linked immunosorbent assay (ELISA) determination of blood sampling time and blood sample ACLY

The patient enters the PICU to collect peripheral blood and obtain serum for later use. Serum ACLY levels were measured by enzyme-linked immunosorbent assay (ELISA) (allied biotechnology limited, hangzhou).

5. Other index Collection

Clinical parameters such as age, sex, underlying disease, source of infection, PRISM III score, mechanical ventilation or vasoactive drug support were collected according to a pre-established Case Report Form (CRF). Laboratory indices include lactic acid (Lac), blood routine (platelets, PLT); the percentage of lymphocytes, monocytes, natural killer [ NK ] cells, T cells or B cells and the infection index (C-reactive protein [ CRP ], leukocytes [ WBC ], interleukin-6 [ IL-6], tumor necrosis factor-alpha (TNF-alpha) the outcome variables are PICU hospitalization time and discharge survival.

Statistical analysis

Data analysis used STATA 15.0MP (College Station, Texas, USA). And evaluating the relation between the serum ACLY level and the PICU mortality by adopting multifactorial logistic regression analysis, and analyzing the correlation between the serum ACLY level and immune cell proportion and inflammatory factor proportion. The receiver operating characteristic curve (ROC) was used to evaluate the clinical value of serum ACLY in identifying the onset of sepsis in children and prognostic evaluation. P <0.05 is statistically significant for the differences.

II, experimental results:

1. mouse serum proteomics identification finds that ACLY exists in serum

After LPS treatment of mice, ACLY was identified in mouse sera by mass spectrometry (fig. 1).

2. LPS causes the increase of the serum ACLY level of the mice, LPS is injected into the abdominal cavity of the mice to prepare endotoxemia, and the serum ACLY level is obviously increased (figure 2)

3. The serum ACLY level of children sepsis infant is obviously higher than that of healthy children

Serum levels of ACLY were significantly elevated in sepsis patients relative to serum levels of ACLY in 30 healthy children (fig. 3, P < 0.001).

4. Serum ACLY has better efficacy on identifying sepsis

The area under the ROC curve for serum ACLY diagnosis of sepsis recognition when the infant was admitted to the PICU was 0.855 (95% CI:0.757-0.952) (FIG. 4). When the Cutoff value of the serum ACLY level is 9.96ng/mL, the sensitivity of the sepsis diagnosis is 100 percent, and the specificity is 72.4 percent; the increase of the serum ACLY level is prompted, and the clinical application value for early warning the generation of sepsis is realized.

5. Relative reduction in serum ACLY levels in children with sepsis

Relative to surviving sepsis patients, serum ACLY levels were significantly lower when dead patients entered the PICU (P ═ 0.014) (fig. 5). Whether a lower serum ACLY level represents a lower starting immunological activity in the infant patient is unknown.

6. Serum ACLY has better efficacy on evaluating sepsis infant prognosis

Evaluation of serum ACLY at PICU the area under the ROC curve for prognosis of sepsis infant was 0.770 (95% CI:0.626-0.915), and when the Cutoff value at the serum ACLY level was 21ng/mL, the sensitivity for diagnosing sepsis was 87.5% and the specificity was 67.6% (FIG. 6); the serum ACLY level is prompted to have certain clinical application value for evaluating the sepsis prognosis.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

Use of a quantitative detection agent for ATP citrate lyase for the manufacture of a kit for the diagnostic, auxiliary diagnostic or prognostic analysis of sepsis.
2. The use of claim 1, wherein the quantitative detection reagent is used to perform any one of the following methods:

the method comprises the following steps of biological mass spectrometry, electrophoresis, chromatography, enzyme-linked immunosorbent assay, immunofluorescence, immunochemiluminescence, immunoturbidimetry, immunoblotting and dot blotting.
3. The use of claim 1, wherein the quantitative detection agent is an antibody specific for ATP citrate lyase.
4. The use according to claim 3, wherein the specific antibody is a monoclonal antibody or a polyclonal antibody.
5. The use of claim 1, wherein the kit further comprises sample treatment reagents comprising at least one of sample lysis reagents, sample purification reagents, and sample nucleic acid extraction reagents.
6. The use according to claim 5, wherein the sample is selected from at least one of blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate, semen and saliva samples of the subject.
7. The use according to any one of claims 1 to 6, wherein the kit further comprises at least one additional reagent for use in the diagnosis, auxiliary diagnosis or prognostic assay of sepsis.
8. Use according to claim 7, wherein the reagent is selected from the group for detecting at least one of:

lactic acid, C-reactive protein, interleukin-6, tumor necrosis factor-alpha, PCT, sICAM-1, supAR, plasma circulating DNA, bacterial DNA, and miRNA.
9. The use according to any one of claims 1 to 6, wherein the reagent is used for blood culture or blood routine testing.
10. Use according to any one of claims 1 to 6, wherein the sepsis is childhood sepsis.