CN112326948B - Biomarker for predicting diabetes, kit and using method thereof - Google Patents

Abstract

The invention discloses a biomarker for predicting diabetes mellitus, a kit and a using method thereof, wherein the biomarker comprises the following bile acid classification indexes: primary free bile acids CA, CDCA, primary conjugated bile acid GCA, GCDCA, TCA, TCDCA, GCDCS, TCDCS, GCDCA _glucuronide, taurine conjugated bile acid TCA, TCDCA, TCDCS, TDCS, TDCA, TLCA, TLCAS, TUDCA and glycine conjugated bile acid GCA, GCDCA, GCDCS, GCDCA _ glucuronide, GDCS, GDCA, GDCA _ glucuronide, GLCA, GLCAS, GUDCA, and the bile acid classification index is used for predicting diabetes with high sensitivity, low cost and good repeatability.

Description

Biomarker for predicting diabetes, kit and using method thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to a biomarker for predicting diabetes mellitus, a kit and a using method thereof.

Background

Diabetes Mellitus (DM) is a metabolic disease caused by impaired insulin secretion or defective action, and is clinically characterized by chronic hyperglycemia in a long period, and is one of the most common chronic diseases in the world. Along with the development of global economy and the continuous improvement of domestic living standard of people, the disease and incidence rate of diabetes rise year by year, and the diabetes becomes a significant factor affecting the healthy life of human beings. In addition, the incidence of diabetic complications is also generally high, including cardiovascular diseases, diabetic nephropathy, fundus diseases, peripheral neuropathy and the like, and is also an important cause of high mortality of diabetics.

At present, the diagnosis of diabetes mainly depends on the levels of plasma glucose and glycosylated hemoglobin, but because of a plurality of influencing factors in the blood glucose detection process, the diagnosis is easily influenced by a plurality of factors such as medicines, diet, emotion and the like, and has certain limitation and has room for improvement. In addition, because diabetes mellitus is hidden, most of the diabetes mellitus lacks or has no typical clinical symptoms, other complications are often found and are combined, and the treatment and prognosis of the diabetes mellitus are seriously influenced. Therefore, the development of a novel detection method with potential to predict or diagnose early-stage diabetes mellitus occurrence is of great significance in reducing the morbidity and even mortality of diabetes mellitus.

In recent years, metabonomics technology has been developed rapidly in the field of diabetes, and main research means include nuclear magnetic resonance, mass spectrometry, chromatography and chromatography-mass spectrometry, and the like, and bile acid histology is a related technical means utilizing metabonomics to explore the prediction effect and correlation of bile acid on diabetes. Marlene Wewalk et al show that compared with normal control population, serum bile acid level of diabetes patients is obviously increased, and bile acid has certain prediction potential for diabetes. At present, no research report of applying bile acid classification indexes to detection of new diabetes exists.

Disclosure of Invention

The invention aims to provide application of bile acid classification indexes in preparing detection reagents or detection objects for predicting diabetes mellitus, and provides a novel auxiliary detection method for clinically screening early diabetes mellitus.

The above object of the present invention is achieved by the following technical solutions:

the present invention provides in a first aspect a biomarker for predicting diabetes comprising at least one bile acid classification indicator, wherein at least one of the bile acid classification indicators is differentially expressed in serum of a subject as compared to a control sample.

Some embodiments of the biomarkers of the invention, the bile acid classification index is selected from one or more of primary free bile acid (Total primary unconjugated BAs), primary conjugated bile acid (Total primary conjugated BAs), taurine conjugated bile acid (Total conjugated BAs) or glycine conjugated bile acid (Total glycone conjugated BAs); wherein:

the primary free bile acid is selected from Cholic Acid (CA) and/or chenodeoxycholic acid (Chenodeoxycholic acid, CDCA);

the primary conjugated bile acid is selected from the group consisting of: glycocholic acid (GCA), glycochenodeoxycholic acid (Glycochenodeoxycholic acid, GCDCA), taurocholic acid (TCA), taurochenodeoxycholic acid (Taurochenodeoxycholic acid, TCDCA), sulfated glycochenodeoxycholic acid (Sulfated glycodeoxycholic acid, GCDCS), sulfated taurochenodeoxycholic acid (Sulfated taurodeoxycholic acid, TCDCS), glucuronidated glycochenodeoxycholic acid (glycochenodeoxycholic acid-glucoronide, gcdca_glucoronide);

the taurine-binding bile acid is selected from the group consisting of: taurocholic acid (TCA), taurocholic acid (Taurochenodeoxycholic acid, TCDCA), sulfated taurochenodeoxycholic acid (Sulfated taurodeoxycholic acid, TCDCS), sulfated taurodeoxycholic acid (Sulfated taurodeoxycholic acid, TDCS), taurodeoxycholic acid (Taurodeoxycholic acid, TDCA), taurocholic acid (Taurolithocholic acid, TLCA), sulfated Niu Huangdan cholic acid (Sulfated taurolithocholic acid, TLCAs), taurocursodeoxycholic acid (Tauroursodeoxycholic acid, TUDCA);

the glycine-binding bile acid is selected from the group consisting of: glycocholic acid (GCA), glycochenodeoxycholic acid (Glycochenodeoxycholic acid, GCDCA), sulfated glycochenodeoxycholic acid (Sulfated glycodeoxycholic acid, GCDCS), glucuronidated glycochenodeoxycholic acid (glycochenodeoxycholic acid-gluconide, gcdca_gluconide), sulfated glycodeoxycholic acid (Sulfated glycodeoxycholic acid, GDCS), glycodeoxycholic acid (Glycodeoxycholic acid, GDCA), glucuronidated glycodeoxycholic acid (Glycodeoxycholic acid-gluconide, gdca_gluconide), glycocholic acid (Glycolithocholic acid, GLCA), sulfated Gan Andan cholic acid (Sulfated glycolithocholic acid, GLCAs), glycoursodeoxycholic acid (Glycoursodeoxycholic acid, GUDCA).

In some embodiments of the biomarkers of the invention, the bile acid classification index may be used in combination with a clinical index for diabetes prediction in a subject.

In some embodiments of the biomarkers of the invention, the diabetes is type 2 diabetes.

In a second aspect, the invention provides a kit for predicting diabetes comprising any one of the biomarkers described above.

In some embodiments of the kit of the invention, the biomarker comprises at least one bile acid classification indicator, and the bile acid classification indicator is selected from one or a combination of two or more of primary free bile acid, primary conjugated bile acid, taurine conjugated bile acid, or glycine conjugated bile acid; wherein:

the primary free bile acid is cholic acid and/or chenodeoxycholic acid;

the primary conjugated bile acid is selected from the group consisting of: glycocholic acid, glycochenodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, sulfated glycochenodeoxycholic acid, sulfated taurochenodeoxycholic acid, glucuronidated glycochenodeoxycholic acid;

the taurine-binding bile acid is selected from the group consisting of: taurocholic acid, tauchenodeoxycholic acid, sulfated taurodeoxycholic acid, taurocholic acid, sulfated Niu Huangdan cholic acid, taurochenodeoxycholic acid;

the glycine-binding bile acid is selected from the group consisting of: glycocholic acid, glycochenodeoxycholic acid, sulfated glycochenodeoxycholic acid, glucuronidated glycochenodeoxycholic acid, sulfated glycodeoxycholic acid, glucuronidated glycodeoxycholic acid, glycolithocholic acid, sulfated Gan Andan cholic acid, glycoursodeoxycholic acid.

In a preferred embodiment of the kit of the present invention, the kit comprises bile acid classification indicators of primary free bile acid, primary conjugated bile acid, taurine conjugated bile acid and glycine conjugated bile acid as qualitative standards for predicting the above-mentioned corresponding bile acid classification indicators in serum of a subject; wherein:

the primary free bile acid comprises both cholic acid and chenodeoxycholic acid;

the primary conjugated bile acid comprises glycocholic acid, glycochenodeoxycholic acid, taurochenoxycholic acid, sulfated glycochenodeoxycholic acid, sulfated taurochenodeoxycholic acid and glucuronidated glycochenodeoxycholic acid simultaneously;

the taurine-conjugated bile acid comprises taurocholic acid, taurochenodeoxycholic acid, sulfated taurodeoxycholic acid, taurocholic acid, sulfated Niu Huangdan cholic acid and tauroursodeoxycholic acid;

the glycine-conjugated bile acid comprises glycocholic acid, glycochenodeoxycholic acid, sulfated glycochenodeoxycholic acid, glucuronidated glycochenodeoxycholic acid, sulfated glycodeoxycholic acid, glucuronidated glycodeoxycholic acid, glycolithocholic acid, sulfated Gan Andan cholic acid, and glycoursodeoxycholic acid.

The invention provides a method for using any kit in a third aspect, wherein the concentration level of the bile acid classification index in the serum of a subject is measured by any kit, then an ROC model is constructed according to the concentration level and is subjected to statistical analysis, and diabetes mellitus of the subject is predicted according to the obtained area under an ROC curve.

Some embodiments of the methods of use of the present invention can be used as an indicator of a subject's risk for developing type 2 diabetes when the area under the ROC curve is > 0.7.

Compared with the prior art, the invention has the beneficial effects that: according to the prediction effect and correlation of bile acid on diabetes mellitus, the invention takes the bile acid classification indexes such as primary free bile acid, primary conjugated bile acid, taurine conjugated bile acid, glycine conjugated bile acid and the like as biological markers for predicting diabetes mellitus, and can judge whether diabetes mellitus risk exists or not by detecting the concentration level of the bile acid in serum of a subject, so that the invention has the advantages of high detection sensitivity, low cost and good repeatability, and provides a novel auxiliary detection method and theoretical basis for clinically screening early diabetes mellitus.

It should be understood that all combinations of the foregoing concepts, as well as additional concepts described in more detail below, may be considered a part of the inventive subject matter of the present disclosure as long as such concepts are not mutually inconsistent.

The foregoing and other aspects, embodiments, and features of the present teachings will be more fully understood from the following description, taken together with the accompanying drawings. Other additional aspects of the invention, such as features and/or advantages of the exemplary embodiments, will be apparent from the description which follows, or may be learned by practice of the embodiments according to the teachings of the invention.

Drawings

FIG. 1 is a ROC graph of bile acid classification index for predicting new onset diabetes in example 1.

FIG. 2 is a graph of ROC demonstrating group-Winzhou center bile acid classification indicators for predicting new onset diabetes; (B) Verifying ROC curve graphs of the group Guiyang center bile acid classification indexes for predicting new diabetes mellitus; (C) Verifying ROC curve graphs for predicting new diabetes by using Shandong province center bile acid classification indexes; (D) Verifying ROC curve graphs of central bile acid classification indexes of Guangzhou mountain for predicting new diabetes; (E) Verifying the ROC curve graph of the group Zhengzhou center bile acid classification index for predicting the new-onset diabetes; (F) And (3) verifying the ROC curve graph of the group Harbin center bile acid classification index for predicting the new-onset diabetes.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without creative efforts, based on the described embodiments of the present invention fall within the protection scope of the present invention. Unless defined otherwise, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.

The terms "first," "second," and the like in the description and in the claims, are not used for any order, quantity, or importance, but are used for distinguishing between different elements. Also, unless the context clearly indicates otherwise, singular forms "a," "an," or "the" and similar terms do not denote a limitation of quantity, but rather denote the presence of at least one. The terms "comprises," "comprising," or the like are intended to cover a feature, integer, step, operation, element, and/or component recited as being present in the element or article that "comprises" or "comprising" does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

The invention is further described with reference to the accompanying drawings and specific examples:

definition of diabetes: fasting glycemia (FBG) not less than 126mg/dL or postprandial glycemia (2-h BG) not less than 200mg/dL, or diabetes mellitus has been diagnosed and hypoglycemic drugs are being taken by a specialist before self-report of a study subject.

Example 1

From the discovery group (n=122), different bile acids in serum are detected by analyzing the different bile acids in the serum through electrospray ionization (ESI) source negative ion mode by using an shimeji nexera x2 ultra-high performance liquid chromatography and shimeji triple quadrupole 8050 mass spectrometer, and the following bile acid classification indexes and the occurrence of diabetes are found to have obvious statistical significance through correction of relevant confounding factors (including age, BMI, fasting blood glucose, smoking and drinking states, diabetes family history, education level, physical activity and the like):

primary free bile acid: CA. CDCA;

primary binding bile acid: GCA, GCDCA, TCA, TCDCA, GCDCS, TCDCS, GCDCA _glucuronide;

taurine binds to bile acids: TCA, TCDCA, TCDCS, TDCS, TDCA, TLCA, TLCAS, TUDCA;

glycine-binding bile acid: GCA, GCDCA, GCDCS, GCDCA _ glucuronide, GDCS, GDCA, GDCA _ glucuronide, GLCA, GLCAS, GUDCA.

The above statistics were validated in 6 validation center (n=810) populations (wenzhou, guiyang, shandong, guangdong, zheng state and harbin) from different provinces across the country, and the above bile acid classification indicators were found to have significant relevance to the risk of developing diabetes, as shown in fig. 2.

In the ROC model, it was shown that the predictive sensitivity and specificity for type 2 diabetes mellitus was significantly increased when the above bile acid classification index was added, as compared to the conventional diagnostic markers fasting glycemia (FBG) and postprandial 2 hours glycemia (2-h BG), with an increase in area under the curve from 0.7713 to 0.8060, as shown in fig. 1.

The specific operation process is as follows:

(1) Serum sample collection and processing

All volunteers enrolled in the study signed informed consent prior to serum sample collection. Blood samples of 122 cases (discovery group) and 810 cases (verification group) of study subjects were collected under the same conditions, and after collection, serum was directly taken after 60 minutes of standing, and stored in a refrigerator at-80 ℃ for later use.

(2) Study object

Using the case control study method, the group was found to incorporate 122 standard-compliant baseline glucose tolerance Normal (NGR) individuals from the nationally cohort 4C study population, including 61 new diabetic individuals and 61 NGR individuals following the follow-up. The validation group included a total of 810 baseline NGR from the 4C cohort population, including 405 new diabetics and 405 NGR individuals following the visit. The inclusion criteria were: 1) The age of the study subjects is greater than or equal to 40 years, 2) the study subjects are subjected to an Oral Glucose Tolerance Test (OGTT); exclusion criteria: at baseline, diabetes or impaired glucose regulation has been experienced. In addition, all subjects received standard questionnaires and physical examinations.

FBG is detected by adopting a fasting venous plasma specimen, and 2-h BG is detected by adopting an OGTT-2h venous plasma specimen. The concentrations of FBG and 2-h BG were measured using an ADVIA-1650 chemoautoanalyzer (Bayer Diagnostics, tarrytown, NY, USA).

Table 1: risk of developing diabetes per elevation of SD level of bile acid classification index

	OR(95％CI)	P value
			Total primary unconjugated	0.90(0.83-0.98)	0.0160
Total primary conjugated	1.13(1.04-1.23)	0.0060
			Total taurine-conjugated BAs	1.10(1.01-1.20)	0.0221
Total glycine-conjugated BAs	1.12(1.03-1.22)	0.0072

The results in table 1 show that per elevated SD level of primary-conjugated bile acids, the risk of developing diabetes is increased by 13% compared to NGR; the risk of developing diabetes increases by 10% per elevated SD level of taurine-conjugated bile acids; the risk of developing diabetes increases by 12% per elevated SD level of glycine-conjugated bile acids.

(3) Serum sample pretreatment

Taking 50 mu L of blood sample, adding 200 mu L of acetonitrile containing internal standard (the internal standard adopted by each compound and the concentration of each internal standard are shown in table 1), extracting target compound, precipitating protein, vortex, centrifuging, sucking 200 mu L of supernatant, freeze-drying, re-dissolving with 50 mu L of aqueous solution containing 25% acetonitrile, and waiting for sample injection.

(4) Bile acid histology analysis

The liquid phase instrument is Shimadzu nexera x2 ultra-high performance liquid chromatography, the mass spectrometer is Shimadzu triple quaternary rod 8050, and a electrospray ionization (ESI) source anion mode is adopted for sample analysis. The main parameters include: nebulizing gas flow3L/min, 10L/min of patterning gas flow, interface temperature ℃, DL temperature 250 ℃, heat block temperature ℃, and 10L/min of patterning gas flow. Multiple Reaction Monitoring (MRM) is used to detect each bile acid result.

The liquid phase separation conditions for sample collection are as follows: the column was 100mm× 2.1mm ACQUITY UPLC C8, particle size 1.7 μm (Waters, USA), phase a contained 10mM ammonium bicarbonate aqueous solution and phase B was pure acetonitrile. Initially 25% phase b, for 0.5 min. Then linearly raised to 40% b in 12.5 minutes. Then, the mixture was raised to 90% B in 1min, rinsed for 3min, and then returned to the original mobile phase in 0.5min, equilibrated for 2.5 min, at a flow rate of 0.35ml/min, column temperature of 35℃and sample injection volume of 5. Mu.L.

After risk assessment of measured bile acids against newly developed type 2 diabetes using a multivariate Logistic regression equation, it was found that after correction of age, body mass index, smoking, drinking, physical activity, education level, diabetes family history and FBG, bile acid classification index was significantly correlated with increased risk of developing type 2 diabetes: primary conjugated bile acid, taurine conjugated bile acid and glycine conjugated bile acid.

The data statistics software SAS was further used to determine the effect of primary free bile acid, primary conjugated bile acid, taurine conjugated bile acid and glycine conjugated bile acid on type 2 diabetes by ROC curve as bile acid classification index, and the results are shown in fig. 1 and 2.

In fig. 1, the group results were found to show that when the classification index was used for predictive prediction of diabetes, the area under ROC curve value was increased to 0.8060, as compared with the area under ROC curve 0.7713 of FBG and 2-hbg for predicting the risk of diabetes occurrence.

In fig. 2A, the validated group wenzhou center results show that the area under ROC curve for classification index used to predict new onset diabetes increases to 0.8444 compared to the area under ROC curve 0.8240 for FBG and 2-hbg used to predict new onset diabetes.

In fig. 2B, the area under the ROC curve at the center of the noble in the validation set increased from 0.7311 to 0.7908.

In fig. 2C, the area under the ROC curve at the center of the provincial center of shandong in the validation set increases from 0.7221 to 0.7531.

In fig. 2D, the area under the ROC curve at the center of the middle mountain in guangzhou proving group increased from 0.6910 to 0.7153.

In fig. 2E, the area under the ROC curve at the center of the state of the validation set increased from 0.6744 to 0.7196.

In fig. 2F, the area under the ROC curve at the center of the haemarkat is verified to increase from 0.6902 to 0.7232.

The results show that the bile acid classification index has better diabetes mellitus prediction potential and has better synergistic effect with clinical diagnosis indexes FBG and 2-hBG. Therefore, the bile acid classification index provided by the invention can be used as a novel serum marker of type 2 diabetes, is used for early screening and diagnosis of diabetes, and provides an auxiliary detection way for clinically evaluating the occurrence risk of diabetes.

The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art will appreciate that, in light of the principles of the present invention, improvements and modifications can be made without departing from the scope of the invention.

Claims (4)

1. The use of a biomarker in the manufacture of a kit for predicting type 2 diabetes, characterized in that,

the biomarker is a bile acid classification index which is differentially expressed in serum of a subject and a control sample, and the bile acid classification index is composed of primary free bile acid, primary conjugated bile acid, taurine conjugated bile acid and glycine conjugated bile acid, wherein:

the primary free bile acid consists of cholic acid and chenodeoxycholic acid;

the primary combined bile acid consists of glycocholic acid, glycochenodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, sulfated glycochenodeoxycholic acid, sulfated taurochenodeoxycholic acid and glucuronidated glycochenodeoxycholic acid;

the taurine-conjugated bile acid consists of taurocholic acid, taurochenodeoxycholic acid, sulfated taurodeoxycholic acid, taurocholic acid, sulfated Niu Huangdan cholic acid and tauroursodeoxycholic acid;

the glycine-conjugated bile acid is composed of glycocholic acid, glycochenodeoxycholic acid, sulfated glycochenodeoxycholic acid, glucuronidated glycochenodeoxycholic acid, sulfated glycodeoxycholic acid, glucuronidated glycodeoxycholic acid, glycolithocholic acid, sulfated Gan Andan cholic acid and glycoursodeoxycholic acid.

2. The use according to claim 1, wherein the bile acid classification index is used in combination with clinical indices for the prediction of type 2 diabetes in a subject, the clinical indices being FBG and 2-hbg.

3. The use according to claim 2, wherein the concentration level of the bile acid classification index in the serum of the subject is determined by the kit, and an ROC model is constructed in combination with clinical indexes, and statistically analyzed, and diabetes of the subject is predicted based on the area under the ROC curve obtained; when the area under the ROC curve is > 0.7, it is used as an indicator of the risk of a subject for type 2 diabetes.

4. A kit for predicting diabetes, characterized in that the kit comprises bile acid classification indicators consisting of primary free bile acid, primary conjugated bile acid, taurine conjugated bile acid and glycine conjugated bile acid as qualitative standards for predicting the above bile acid classification indicators in the serum of a subject, wherein:

the primary free bile acid consists of cholic acid and chenodeoxycholic acid;

the primary combined bile acid consists of glycocholic acid, glycochenodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, sulfated glycochenodeoxycholic acid, sulfated taurochenodeoxycholic acid and glucuronidated glycochenodeoxycholic acid;

the taurine-conjugated bile acid consists of taurocholic acid, taurochenodeoxycholic acid, sulfated taurodeoxycholic acid, taurocholic acid, sulfated Niu Huangdan cholic acid and tauroursodeoxycholic acid;

the glycine-conjugated bile acid is composed of glycocholic acid, glycochenodeoxycholic acid, sulfated glycochenodeoxycholic acid, glucuronidated glycochenodeoxycholic acid, sulfated glycodeoxycholic acid, glucuronidated glycodeoxycholic acid, glycolithocholic acid, sulfated Gan Andan cholic acid and glycoursodeoxycholic acid.