CN108572251B

CN108572251B - Small molecule marker for early stage of cirrhosis and application thereof

Info

Publication number: CN108572251B
Application number: CN201810548561.3A
Authority: CN
Inventors: 李功华; 黄京飞; 李文兴; 代绍兴; 唐承薇; 童欢
Original assignee: Kunming Institute of Zoology of CAS
Current assignee: Lianpu Bing (Chongqing) Pharmaceutical Technology Co.,Ltd.
Priority date: 2018-05-31
Filing date: 2018-05-31
Publication date: 2020-01-17
Anticipated expiration: 2038-05-31
Also published as: CN108572251A

Abstract

The invention provides application of a brand-new liver cirrhosis diagnosis marker in preparation of an early liver cirrhosis diagnosis product, and the marker can be used for performing early cirrhosis diagnosis by single or combination of two or more than two. The liver cirrhosis markers provided by the invention are 20 in total, have the characteristics of high accuracy and high sensitivity, wherein 7 markers, namely pyroglutamic acid, malic acid, octyl glucoside, 3-aminobutyric acid, methylcrotonylglycine, cyclohexenetetraol beta epoxide and ribose, have the area AUC under the characteristic curve line of 1.0, and prompt that the accuracy and the sensitivity of the markers are both more than 99 percent, and can be directly used as the indexes of liver cirrhosis.

Description

Small molecule marker for early stage of cirrhosis and application thereof

Technical Field

The invention relates to the field of biological medicines, in particular to an early diagnosis marker for cirrhosis and application thereof.

Background

China is a country with multiple liver cirrhosis. Liver cirrhosis is mainly caused by alcoholic fatty liver, viral hepatitis, and is characterized by extensive hepatocyte necrosis, nodular regeneration of residual hepatocytes, connective tissue hyperplasia and fibrosepta formation in histopathology. The currently clinically adopted grading standard for cirrhosis is Child-Pugh. In general, the compensatory phase of cirrhosis belongs to the Child-Pugh grade A. Because of the strong liver compensation function, patients with cirrhosis in this stage often have no obvious symptoms. While the decompensation stage of cirrhosis belongs to Child-Pugh grade B and C, and patients in the stage can have symptoms related to liver damage, mainly manifested as portal hypertension and complicated with multiple system involvement. Complications such as upper gastrointestinal hemorrhage, Hepatic Encephalopathy (HE), secondary infection, splenic hyperfunction, ascites, canceration and the like often appear in the late stage of cirrhosis, and the health of human beings is seriously affected.

At present, no good treatment means exists for the advanced stage of cirrhosis, namely the decompensation stage, for example, the 1-year fatality rate of Hepatic Encephalopathy (HE) caused by late cirrhosis is more than 64%, and the fatality rate of functional renal failure (HRS) caused by late cirrhosis is more than 95%. In contrast to the advanced stage, patients with early stage cirrhosis can effectively prevent the malignant development of cirrhosis or reverse liver injury to achieve the effect of long-term survival and even cure through etiological treatment (such as hepatitis B virus treatment), lifestyle improvement (such as early stage cirrhosis caused by alcoholic fatty liver) and the like. Therefore, the early discovery and prevention of liver cirrhosis, the prevention of disease progression, and the avoidance of clinical decompensated complications are the basic principles of liver cirrhosis treatment. However, liver has strong compensatory ability, early cirrhosis does not show obvious clinical symptoms, and when the symptoms are obvious, the late cirrhosis is achieved. Therefore, the discovery of the diagnosis marker of early cirrhosis has good clinical significance and application value.

The means used clinically to confirm cirrhosis of the liver relies primarily on ultrasound imaging and confirmation by liver puncture. The sensitivity of ultrasonic diagnosis is low, and liver puncture damages the liver of a patient, so that the risk exists, the popularization is not easy, and many patients cannot be diagnosed until the decompensation period of cirrhosis. Recently, studies show that serum HA, CG, PIII NP, PC III, CIV, LN and the like are increased in liver cirrhosis patients, but the sensitivity and the specificity of the serum are to be improved, so that the serum is not clinically adopted as an index of early-stage liver cirrhosis at present. Therefore, the discovery of new early stage liver cirrhosis markers is urgently needed.

Disclosure of Invention

The invention provides a brand-new liver cirrhosis diagnosis marker, and the marker can be used for early diagnosis of liver cirrhosis by single or combination of two or more.

The invention provides 20 liver cirrhosis markers which comprise: 5-Methylthioadenosine (5-Methylthioadenosine, abbreviated as MTA), 3-Hydroxybutyric acid (abbreviated as bhb), Pyroglutamic acid (Pyroglutamic acid), Glucose 1-phosphate (Glucose-1-phosphate), Malic acid (L-maleic acid), Octylglucoside (beta-Octylglucoside), 3-aminobutyric acid (3-Aminobutanoic acid), Thromboxane B2(Thromboxane B2), Allopurinol nucleoside (Allopurinol riboside), methylcrotonylglycine (tiglyglycine), taurocholic acid (Taurocholate), galactaric acid (galactaric acid), Sedoheptulose (Sedoheptulose), D-galacturonic acid (D-galatoside), cyclohexenetetraol β epoxide (galacturonic acid B), heptadecanoic acid (heptadecanoic acid), arachidonic acid (arachidonic acid), and so on.

Of the 20 cirrhosis markers, the significant increase in cirrhosis patients was: 11 kinds of 5-methylthioadenosine, 3-hydroxybutyric acid, pyroglutamic acid, glucose-1-phosphate, octyl glucoside, 3-aminobutyric acid, taurocholic acid, galactonic acid, sedoheptulose, D-galacturonic acid and cyclohexenetetraol beta-epoxide.

Of the 20 cirrhosis markers, significantly reduced in patients with cirrhosis were: malic acid, thromboxane B2, allopurinol nucleoside, methylcrotonylglycine, heptadecanoic acid, arachidonic acid, pyruvic acid, inosine, and ribose.

The invention also provides application of the 20 liver cirrhosis markers in preparing an early liver cirrhosis diagnosis product, which is characterized in that the diagnosis product comprises a reagent for detecting the one or more markers, and the reagent is used for judging whether the subject has early liver cirrhosis or not by detecting the concentration of the one or more markers in body fluid of the subject.

Preferably, the marker is pyroglutamic acid, malic acid, octyl glucoside, 3-aminobutyric acid, methylcrotonylglycine, cyclohexatetraol beta epoxide, ribose.

Further, the body fluid is serum or plasma.

Further, the reagent is a reagent required for detecting the concentration of the one or more compounds based on a chemical analysis method or a mass spectrometry method.

Further, if the concentration of the marker 5-methylthioadenosine and/or 3-hydroxybutyric acid and/or pyroglutamic acid and/or 1-phosphoglucose and/or octylglucoside and/or 3-aminobutyric acid and/or taurocholic acid and/or galactaric acid and/or sedoheptulose and/or D-galacturonic acid and/or cyclohexatetraol beta epoxide is significantly increased compared to the normal standard concentration, the subject is judged to have early cirrhosis.

Further, if the concentration of the marker malic acid and/or thromboxane B2 and/or allopurinol nucleoside and/or methylcrotoylglycine and/or heptadecanoic acid and/or arachidonic acid and/or pyruvic acid and/or inosine and/or ribose is significantly reduced compared to the normal standard concentration, the subject is judged to have early cirrhosis.

Further, the diagnostic product is a kit, chip or assay platform.

The accuracy of markers used in the compositions and methods of the invention can be characterized by plotting a characteristic curve ("ROC curve"). ROC is a plot of true positive rate versus false positive rate for different possible cut points of a diagnostic marker. The ROC curve shows the relationship between sensitivity and specificity. That is, an increase in sensitivity will be accompanied by a decrease in specificity. The closer the curve is to the left axis of the ROC space, then the top edge, the more accurate the marker. Conversely, the closer the curve is to the 45 degree diagonal of the ROC curve, the less accurate the marker. The area under ROC (referred to as "AUC") is a measure of marker accuracy. The accuracy of the markers depends on how well the markers properly classify the components to be tested into groups with the disease and groups without the disease. An AUC of 1 indicates a perfect marker, while an area of 0.5 indicates a less useful marker.

Through prospective study on the sera of 10 cirrhosis patients and 9 normal patients, the change of each marker in cirrhosis patients and the corresponding area under the characteristic curve AUC are shown in Table 1.

TABLE 1 Change in the liver cirrhosis patients for each marker and the corresponding area under the characteristic Curve AUC

As can be seen from table 1, the area under the characteristic curve AUC of all 20 markers is greater than 0.88, and the area under the characteristic curve AUC of 7 markers is 1. The AUC value indicates the predictive ability of the marker, and the AUC 1 indicates 100% accuracy in identifying early cirrhosis. Therefore, it can be seen from table 1 that the early diagnosis marker for cirrhosis provided by the present patent has the characteristics of high accuracy and high sensitivity.

The detailed changes of the 20 liver cirrhosis markers in the blood serum of healthy volunteers and liver cirrhosis patients are shown in 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A, 16A, 17A, 18A, 19A and 20A in the attached figures 1-20.

The corresponding molecular formulas of the 20 liver cirrhosis markers are shown in figures 1-20 as 1B, 2B, 3B, 4B, 5B, 6B, 7B, 8B, 9B, 10B, 11B, 12B, 13B, 14B, 15B, 16B, 17B, 18B, 19B and 20B.

The invention has the beneficial effects that: at present, no better early cirrhosis marker exists, the cirrhosis marker provided by the invention has the characteristics of high accuracy and high sensitivity, wherein 7 markers, namely pyroglutamic acid, malic acid, octyl glucoside, 3-aminobutyric acid, methylcrotonylglycine, cyclohexatetraol beta epoxide and ribose, have the area AUC under a characteristic curve line of 1.0, and prompt that the accuracy and the sensitivity of the marker are both more than 99 percent, and the marker can be directly used as an index of cirrhosis.

Drawings

FIGS. 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A, 16A, 17A, 18A, 19A and 20A are detailed graphs of the changes of the 20 cirrhosis markers in healthy volunteers and cirrhosis patients.

Fig. 1B, 2B, 3B, 4B, 5B, 6B, 7B, 8B, 9B, 10B, 11B, 12B, 13B, 14B, 15B, 16B, 17B, 18B, 19B, and 20B are molecular formulas corresponding to the 20 liver cirrhosis markers.

Detailed Description

The invention is further illustrated by the following examples, which are not to be construed as further limiting. The contents of the present invention can be more easily understood by referring to the following detailed description of the implementation method of the present invention and the included examples. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The first embodiment is as follows: 5-Methylthioadenosine (5-Methylthioadenosine, MTA for short) is used as a single marker for early diagnosis of liver cirrhosis. The specific implementation method comprises the following steps:

1. obtaining a sample of a subject, and carrying out pretreatment: collecting blood sample of a subject according to a standard and reasonable operation process, adding 100 mu L of blood into 350 mu L of methanol, performing vortex mixing, performing high-speed centrifugation, taking supernatant, performing concentration drying, adding 100 mu L of acetonitrile for redissolution, and performing UHPLC-QTOFMS test directly through a filter plug.

2. Main reagent 1) Methanol (Methanol, LOT #1230, LCMS grade, CAS: 67-56-1) from CNWTcologies (ANPEL Laboratory Technologies (Shanghai) Inc.)); acetonitrile (Acetonitrile, LOT #1646, LCMS grade, CAS: 75-05-8) from CNW Technologies (ANPEL laboratory Technologies (Shanghai) Inc.); water (provided by a water purifier); ammonium acetate (Ammonium acetate, LOT #1350K100, LCMS grade, CAS: 631-61-8) was purchased from CNW Technologies (ANPEL laboratory Technologies (Shanghai) Inc.); ammonia (Ammonium hydroxide, LCMS grade, CAS: 1336-21-6) from CNW Technologies (ANPEL Laboratory Technologies (Shanghai) Inc.); l-2-chlorophenylalanine (2-Chloro-L-phenylalanine, CAS:103616-89-3, purity ≥ 98%), available from Hevea championii Biotech, Inc., China.

3. The main instrument platforms used: 1) UHPLC-QTOFMS (Agilent 1290UHPLC + AB tripleTOF 6600); 2) a chromatographic column: waters Acquity UPLC, HSS tj31.8um, 2.1 × 101 mm: 3) a centrifuge: heraeus fresco17centrifuge, Thermo Scientific; 4) vortex appearance: VORTEX-5, Linebel Instrument manufacturing, Inc.; 5) a water purifier: clear D24UV, Merck Millipore, Germany; 6) an ultrasonic instrument: PS-60AL, Shenzhen, Redebang electronics, Inc., China.

4. Data processing: and after the UHPLC-QTOFMS is tested, collecting UHPLC-QTOFMS data for analysis. The data processing uses XCMS program, and the parameter setting refers to software description. After the XCMS program treatment, indexes such as charge-to-mass ratio, retention time, peak area, secondary mass spectrum and the like of all metabolites are obtained.

5. Determination of the concentration of 5-methylthioadenosine: mass-to-charge-mass ratios and mass spectrum secondary fragments are determined by aligning secondary mass spectrum databases. The concentration of methylthioadenosine in blood is determined by the ratio of its peak area to that of the standard.

6. Diagnosis of cirrhosis: the above-measured concentration of 5-methylthioadenosine in the blood of the subject was compared with normal data. If the value exceeds the threshold for a healthy population, then the subject has a higher risk of cirrhosis. Wherein the threshold value of 5-methylthioadenosine in the blood of healthy people is 4.0 nmol/L. The results showed an area of 0.911 under the characteristic curve.

The second embodiment is as follows: 3-Hydroxybutyric acid (bhb for short) is used as a single marker for early diagnosis of liver cirrhosis. The concentration of 3-hydroxybutyrate in the subject was determined in the same manner as in 1-5 steps of the first embodiment. The above-measured 3-hydroxybutyrate concentration in the blood of the subject was compared with normal data. If the value exceeds the threshold for a healthy population, then the subject has a higher risk of cirrhosis. Wherein the threshold value of the 3-hydroxybutyric acid in the blood of healthy people is 100 umol/L. The results showed an area of 0.889 under the characteristic curve.

The third concrete implementation mode: the early diagnosis of cirrhosis is carried out by combining a plurality of metabolites shown in the table I. The concentrations of the various metabolites of the subject are determined in steps 1-5 of the first embodiment. The above measured concentrations of various metabolites in the blood of the subject were compared with normal data. If one or more of the values exceed a threshold for blood in a healthy population, the subject has a higher risk of cirrhosis.

Claims

1. Use of a marker consisting of 5-methylthioadenosine and 3-hydroxybutyric acid and pyroglutamic acid and glucose-1-phosphate and malic acid and octylglucoside and 3-aminobutyric acid and thromboxane B2 and allopurinol nucleoside and methylcrotonylglycine and taurocholic acid and L-galactonic acid and sedoheptulose and D-galacturonic acid and cyclohexatetraol beta epoxide and heptadecanoic acid and arachidonic acid and pyruvic acid and inosine and ribose for the preparation of an early diagnosis product for cirrhosis, characterized in that said diagnosis product comprises a reagent for detecting said marker, said reagent determining whether a subject suffers from early cirrhosis by detecting the concentration of said marker in the body fluid of the subject.

2. Use according to claim 1, wherein the body fluid is serum or plasma.

3. The use according to claim 1, wherein the reagent is a reagent required for detecting the concentration of a marker based on chemical analysis or mass spectrometry.

4. Use according to any one of claims 1 or 2 or 3, characterized in that the concentration of 5-methylthioadenosine and 3-hydroxybutyric acid and pyroglutamic acid and glucose and octylglucoside 1-phosphate and glucose and octylglucoside 3-aminobutanoic acid and taurocholic acid and galactonic acid and sedoheptulose and D-galacturonic acid and cyclohexenetetraol β epoxide in the body fluid of a cirrhosis patient is significantly increased compared to the normal standard concentration.

5. The use as claimed in any one of claims 1 or 2 or 3, wherein the concentration of malic and thromboxane B2 and allopurinol nucleoside and methylcrotonylglycine and heptadecanoic acid and arachidonic acid and pyruvic acid and inosine and ribose in the body fluids of patients with cirrhosis is significantly reduced compared to the normal standard concentration.

6. Use according to any one of claims 1 to 3, wherein the diagnostic product is a kit, chip or assay platform.