Marker combination for diagnosis or condition monitoring of intrahepatic cholestasis of pregnancy and application thereof

Abstract

The present application relates to the technical field of diagnostic markers and diagnostic methods for intrahepatic cholestasis of pregnancy (ICP), and in particular to a combination of diagnostic or condition monitoring markers for intrahepatic cholestasis of pregnancy and application thereof. The biomarkers include diagnostic markers for diagnosis and screening, and condition monitoring markers for follow-up; the diagnostic markers include a diagnostic combination marker composed of tauroursodeoxycholic acid and glycochenodeoxycholic acid, and taurohyocholic acid; and the condition monitoring markers include glycochenodeoxycholic acid, glycolcholic acid, and taurohyocholic acid. The technical solution solves the technical problem of the prior art that there is a lack of sensitive and specific diagnostic markers for intrahepatic cholestasis of pregnancy, and has important significance for early diagnosis, auxiliary clinical diagnosis and treatment, and improvement of population quality, and provides a new strategy for the difficult diagnosis and treatment of ICP.

Description

Technical Field

[0001] This invention relates to the technical field of diagnostic markers and methods for intrahepatic cholestasis of pregnancy, specifically to combinations of bile acid diagnostic or disease monitoring markers for intrahepatic cholestasis of pregnancy and their applications. Background Technology

[0002] Intrahepatic cholestasis of pregnancy (ICP) is a common pregnancy-specific condition characterized by varying degrees of maternal itching, elevated serum bile acids, and / or liver dysfunction in the mid-to-late stages of pregnancy. It primarily affects perinatal outcomes, potentially leading to spontaneous preterm birth, meconium-stained amniotic fluid, fetal distress, and stillbirth. The incidence of ICP is relatively high in Chongqing, Chengdu, and Shanghai in my country, ranging from 3.2% to 6.3%, with a recurrence rate as high as 40% to 70%. ICP has been classified as a high-risk pregnancy and remains a significant challenge for obstetricians and expectant mothers.

[0003] Currently, the diagnosis of ICP is mainly based on the 2015 Chinese Medical Association Obstetrics and Gynecology Branch ICP Diagnosis and Treatment Guidelines: the diagnosis of ICP is mainly based on pruritus symptoms, serum total bile acid (TBA) levels higher than 10 μmol / L and / or elevated serum transaminases (excluding other diseases that cause cholestasis or pruritus), and spontaneous relief of symptoms and signs 4-6 weeks postpartum. TBA is currently the most useful laboratory evidence for diagnosing ICP.

[0004] Early diagnosis, intervention, and appropriate treatment of intravascular coagulation (ICP) can effectively reduce the risk of the disease and maternal and infant complications, significantly alleviating the suffering and economic burden on mothers, infants, and their families. However, current clinical monitoring methods for ICP are very limited, relying primarily on total bile acids (TBA), which have relatively low sensitivity. Previous studies have found that some early-stage ICP patients only exhibit abnormalities in indicators such as alanine, aspartate aminotransferase (AST), bilirubin, or gamma-glutamyl transferase (GGT), and their clinical manifestations are often atypical. Therefore, diagnosing ICP based on TBA alone can lead to a high false-negative rate, resulting in missed diagnoses and delays in optimal treatment. Furthermore, during treatment, some patients may experience a brief period of normal TBA levels followed by a subsequent increase, easily leading to misdiagnosis.

[0005] Therefore, finding a sensitive and specific diagnostic biomarker for ICP is of great significance for achieving accurate diagnosis of ICP and guiding clinical treatment. Summary of the Invention

[0006] The present invention aims to provide a combination of bile acid diagnostic or disease monitoring biomarkers for intrahepatic cholestasis of pregnancy, in order to solve the technical problem of the lack of sensitive and specific diagnostic biomarkers for intrahepatic cholestasis of pregnancy in the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The application of biomarkers in the development of systems for the diagnosis or monitoring of intrahepatic cholestasis of pregnancy, wherein the biomarkers include diagnostic biomarkers for diagnosis and screening; and the diagnostic biomarkers include a combined diagnostic biomarker composed of taurine-ω-mouse cholic acid and glycochenodeoxycholic acid.

[0009] This invention also provides a biomarker detection system for the diagnosis or monitoring of intrahepatic cholestasis of pregnancy, characterized in that it is used to detect the serum concentration of biomarkers; the biomarkers include diagnostic biomarkers for diagnosis and screening and disease monitoring biomarkers for follow-up; the diagnostic biomarkers include a combined diagnostic biomarker composed of taurine-ω-mouse cholic acid and glycochenodeoxycholic acid and / or taurine cholic acid; the disease monitoring biomarkers include at least one of glycochenodeoxycholic acid, glycocholic acid, and taurine cholic acid;

[0010] The marker detection system includes a pretreatment unit, a liquid chromatography unit, and a mass spectrometry unit.

[0011] Furthermore, the serum concentration of the combined diagnostic marker is calculated as follows: 33.152x1 + 2.637x2; where x1 is the serum concentration of taurine-ω-mouse cholic acid and x2 is the serum concentration of glycochenodeoxycholic acid; and the diagnostic threshold of the combined marker is 4.41 μmol / L.

[0012] Furthermore, the diagnostic markers also include taurine.

[0013] Furthermore, the diagnostic threshold for taurine bile acid is 0.0153 μmol / L.

[0014] Furthermore, the diagnostic markers are used for the early diagnosis of intrahepatic cholestasis of pregnancy.

[0015] Furthermore, the biomarkers also include disease detection biomarkers for follow-up; the disease monitoring biomarkers include at least one of glycochenodeoxycholic acid, glycocholic acid, and taurine cholic acid.

[0016] Furthermore, the diagnostic threshold for glycochenodeoxycholic acid is 0.563 μmol / L; the diagnostic threshold for glycocholic acid is 0.608 μmol / L; and the diagnostic threshold for taurine cholic acid is 0.0153 μmol / L.

[0017] Furthermore, the parameters of the liquid chromatography unit are set as follows:

[0018] Chromatographic column: Kinetex XB-C18 column, 100 mm in length, 2.1 mm in diameter, and 2.6 μm particle size of packing material;

[0019] Column temperature: 30℃;

[0020] Mobile phase: Mobile phase A is 15 mmol / L ammonium acetate solution, and mobile phase B is acetonitrile;

[0021] Elution method: gradient elution as set in the program below;

[0022]

[0023]

[0024] Flow rate: 0.4 mL / min;

[0025] Injection volume: 5 μL.

[0026] Using the above chromatographic conditions, the inventors can separate more candidate biomarkers and more accurately reflect the content levels of these candidate biomarkers in serum samples, creating conditions for a more comprehensive screening of ICP biomarkers.

[0027] Furthermore, the parameters of the mass spectrometry unit are set as follows: Ion source: electrospray ion source;

[0028] Mass spectrometry parameter settings: Ion spray voltage: -4500V; Nebulizing gas: 55mL / min; Auxiliary heating gas: 55mL / min; Curtain gas: 25mL / min; Ion source temperature: 550℃;

[0029] A negative ion scanning mode was used for TOF MS first-order mass spectrometry analysis in the range of m / z 70-1000, with a scan time of 500 ms. The precise mass number of each parent ion m / z ± 0.05 Da was used as the target ion, and the daughter ion was scanned in the range of m / z 70-600, with a scan time of 100 ms.

[0030] Furthermore, for taurine-ω-mouse cholic acid and taurine-pork cholic acid, the mother ion m / z is 514.28, the daughter ion m / z includes 79.95, 106.98 and 124.00, the declustering voltage is -60V and the collision energy is -80V; for glycochenodeoxycholic acid, the mother ion m / z is 448.30, the daughter ion m / z is 74.02, the declustering voltage is -60V and the collision energy is -50V; for glycocholic acid, the mother ion m / z is 464.30, the daughter ion m / z is 74.02, the declustering voltage is -50V and the collision energy is -50V.

[0031] In summary, the beneficial effects of this technical solution are as follows:

[0032] (1) Metabolic profiling analysis is a level of metabolomics research. It involves the qualitative and quantitative analysis of all intermediate products or marker components of multiple metabolic pathways of a certain class of structurally and property-related compounds or a certain metabolic pathway. It has higher accuracy than traditional diagnostic methods that rely on a single biomarker. Through the above analysis, the inventors discovered a variety of biomarkers that can be used for ICP diagnosis, including:

[0033] A combined biomarker consisting of taurine-ω-mouse cholic acid and glycochenodeoxycholic acid (T-ω-MCA+GCDCA): This is a novel biomarker combination that can be used for the initial diagnosis and screening of ICP. In particular, when used for initial diagnosis, this biomarker combination can diagnose early-stage ICP patients, significantly advancing the time to diagnosis and enabling treatment of these patients before the appearance of obvious clinical symptoms, effectively improving their pregnancy outcomes and quality of life.

[0034] Taurine bile acid (THCA): It has high specificity for the initial diagnosis and screening of ICP, and can assist in diagnosis and rule out false positives. It can also be used for disease monitoring and follow-up, accurately reflecting the patient's condition.

[0035] Glycine chenodeoxycholic acid (GCDCA): Used for disease monitoring and follow-up, accurately reflecting the patient's condition.

[0036] Glycinecholic acid (GCA): Used for disease monitoring and follow-up, accurately reflecting the patient's condition.

[0037] In pregnant women with intracranial pressure cough (ICP), the complexity and diversity of bile acid metabolism play different roles in the development of ICP, leading to variations in the trends and degrees of bile acid metabolism changes, thus forming a specific bile acid metabolism profile. Through metabolic profile analysis, the biomarkers screened in this technical approach can be used for the accurate diagnosis of ICP.

[0038] (2) Compared with existing technologies, this method optimizes the elution gradient conditions of chromatography, improves the separation of isomers, and achieves effective separation of more bile acids. Ultra-high performance liquid chromatography-high resolution triple quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS / MS) was used to analyze the bile acid metabolism profile of serum from ICP patients, revealing the differences in bile acid metabolism profiles between normal individuals and ICP patients. Multivariate statistical analysis was then used to screen for diagnostic biomarkers for ICP.

[0039] (3) The present invention adds a validation group, with a total sample size of 289 cases in the two embodiments. The large sample size makes the analysis results more reliable. The present invention discloses bile acid diagnostic markers for ICP, which solves the problem of low sensitivity of serum TBA detection for early ICP patients. It can be used for early diagnosis of ICP and to seize the best time for treatment. The bile acid markers disclosed in the present invention can also be used for follow-up of ICP patients during drug treatment, and the results are better than TBA. Attached Figure Description

[0040] Figure 1 The total ion chromatogram is for the mixed bile acid standard of Example 1.

[0041] Figure 2 PLS-DA scores of serum bile acids in ICP patients and normal pregnant women in Example 1 are shown.

[0042] Figure 3 This serves as a permutation verification of the ICP pregnant woman and healthy pregnant woman PLS-DA model in Example 1.

[0043] Figure 4 ROC curves for bile acid markers in the diagnosis of ICP (A: Logistic regression analysis, combined T-ω-MCA and GCDCA; B: THCA). Detailed Implementation

[0044] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0045] The key terms used in this technical solution are defined as follows:

[0046] Early diagnosis of ICP: In the early stages of ICP development, it may not be possible to diagnose ICP using the diagnostic criteria outlined in the "Guidelines for the Diagnosis and Treatment of Intrahepatic Cholestasis of Pregnancy (2015)" issued by the Chinese Society of Obstetrics and Gynecology. However, as the disease progresses, it may eventually be diagnosed as ICP using the same criteria. Early diagnosis of ICP refers to confirming the diagnosis in its early stages (when the above-mentioned standard methods are insufficient).

[0047] Initial diagnosis: First visit to the hospital during pregnancy.

[0048] Follow-up: A method of observation to regularly monitor changes in the condition of ICP patients and guide their recovery after drug treatment.

[0049] Example 1: Profile analysis of bile acid metabolism and screening of potential biomarkers in intrahepatic cholestasis of pregnancy

[0050] Serum samples were collected from newly diagnosed patients with intrahepatic cholestasis of pregnancy (ICP) and healthy pregnant women. After preprocessing, the samples were analyzed by UPLC-QTOF-MS / MS. Partial least squares discriminant analysis (PLS-DA) was used to visualize the differences in bile acid metabolism profiles between ICP patients and healthy pregnant women. Based on variable weights (VIP) values ​​and multivariate statistical analysis, bile acid biomarkers for ICP were screened, providing strong technical support for the accurate diagnosis of ICP.

[0051] (I) Sample situation

[0052] Intrahepatic cholestasis of pregnancy (ICP) is a complication specific to the second and third trimesters of pregnancy, characterized by pruritus and jaundice as the main clinical manifestations, and elevated serum bile acids. A total of 179 ICP patients and normal pregnant women were included in this study (diagnostic criteria were based on the "Guidelines for the Diagnosis and Treatment of Intrahepatic Cholestasis of Pregnancy (2015)" issued by the Chinese Society of Obstetrics and Gynecology). Among them, 91 were in the control group and 88 were ICP patients; details are shown in Table 1.

[0053] Table 1: Liver function tests and pregnancy information in the ICP group and the control group (quantitative data in the table are expressed as M(Q1,Q3)).

[0054]

[0055] (II) Experimental Methods

[0056] (1) Sample preprocessing

[0057] Sample pretreatment was performed using solid-phase extraction (Bond Elute C18 (3 mL / 200 mg, Agilent Technologies, USA)). Serum samples were thawed on ice. 300 μL of serum was taken and 1 mL of ultrapure water and 10 μL of internal standard (20 μmol / L, CDCA-d4, CA-d4, LCA-d4, Shanghai Zhenzhun Biotechnology Co., Ltd.) were added and mixed well. Activation: 1 mL of methanol solution and 1 mL of ultrapure water were added sequentially. Sample loading: The diluted sample was added to a solid-phase extraction column. Eluting: 2 mL of ultrapure water was added, and the sample was dried by gravity dripping and then under pressure. Elution: 1.5 mL of methanol / water (9 / 1, v / v) solution was added, the eluent was collected, dried by gravity dripping and then under pressure. The sample was evaporated to dryness under vacuum at low temperature. The sample was reconstituted with 60 μL of methanol / water (1 / 1, v / v) solution (vortexed for 1 min, sonicated for 1 min), centrifuged for 10 min, and the supernatant (13000×g, 4℃) was collected for UPLC-QTOF-MS / MS detection.

[0058] (2) UPLC-QTOF-MS / MS detection

[0059] The analytical instrument used in this embodiment is the AB SCIEX ExionLC 2.0 ultra-high pressure liquid chromatography system with Triple Time-of-Flight (TOF). TM The 6600 high-resolution mass spectrometry system uses an electrospray ionization source in negative ion mode for sample analysis.

[0060] The chromatographic conditions are as follows:

[0061] Chromatographic column: Kinetex XB-C18 column (100mm×2.1mm, 2.6μm);

[0062] Column temperature: 30℃;

[0063] Mobile phase: Mobile phase A is 15 mmol / L ammonium acetate solution, and mobile phase B is acetonitrile;

[0064] Elution method: Gradient elution (see Table 2 for the gradient elution procedure);

[0065] Flow rate: 0.4 mL / min;

[0066] Injection volume: 5 μL.

[0067] Table 2: Gradient elution program

[0068]

[0069] Mass spectrometry conditions are:

[0070] Ion source: Electrospray ionization (ESI); mass spectrometry parameters were set as follows: ion spray voltage: -4500V; nebulizing gas: 55mL / min; auxiliary heating gas: 55mL / min; curtain gas: 25mL / min; ion source temperature: 550℃. A negative ion scanning mode was used for TOF MS first-order mass spectrometry analysis in the m / z range of 70-1000, with a scan time of 500ms. Target ions were selected based on the precise mass number (m / z ± 0.05 Da) of each precursor ion, and daughter ion scanning was performed in the m / z range of 70-600, with a scan time of 100ms. Characteristic fragment ion information was obtained using the precursor ions as the quantitative ions. The quantitative ion m / z, characteristic daughter ion m / z, declustering voltage, and collision energy for bile acid analysis are shown in Table 3.

[0071] Table 3: Bile Acid Mass Spectrometry Analysis Conditions

[0072]

[0073] The bile acids mentioned above are: ω-mouse cholic acid (ω-MCA), α-mouse cholic acid (α-MCA), porcine cholic acid (HCA), cholic acid (CA), ursodeoxycholic acid (UDCA), porcine deoxycholic acid (HDCA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), taurine-ω-mouse cholic acid (T-ω-MCA), taurine-α-mouse cholic acid (T-α-MCA), taurine porcine cholic acid (THCA), taurine cholic acid (TCA), and taurine ursodeoxycholic acid (TCA). The following bile acids were used: Taurine deoxycholic acid (THDCA), Taurine chenodeoxycholic acid (TCDCA), Taurine deoxycholic acid (TDCA), Taurine lithocholic acid (TLCA), Glycine chenodeoxycholic acid (GHCA), Glycine cholic acid (GCA), Glycine chenodeoxycholic acid (GCDCA), Glycine deoxycholic acid (GDCA), Glycine lithocholic acid (GLCA), Chenodeoxycholic acid-d4 (CDCA-d4), Cholic acid-d4 (CA-d4), and Lithocholic acid-d4 (LCA-d4). All bile acid standards were purchased from Shanghai Zhenzhun Biotechnology Co., Ltd. Ammonium acetate was of mass spectrometry grade, and methanol and acetonitrile were of chromatographic grade.

[0074] (III) Results Analysis

[0075] (1) Mass spectrometry data analysis

[0076] All ICP group and control group samples were analyzed by UPLC-QTOF-MS / MS.

[0077] Bile acids were qualitatively and quantitatively analyzed using PeakView 2.2 and MultiQuant 3.0.3 software (AB SCIEX, USA). Under the same chromatographic and mass spectrometric conditions, standard solutions prepared from mixed bile acid standards and serum samples were analyzed by UPLC-QTOF-MS / MS. The precursor ion was used as the quantitative ion to obtain characteristic fragment ion information. The m / z of the quantitative ion, the m / z of the characteristic fragment ion, the declustering voltage, and the collision energy for bile acid analysis are shown in Table 3. Based on the chromatographic peaks in the serum samples that showed the same retention time as the bile acids in the standard solutions, the peaks were identified as the corresponding bile acid peaks in the serum samples. The total ion chromatogram of the mixed standards is shown in the figure below. Figure 1 As shown.

[0078] 10 μL of a series of mixed standard solutions of varying concentrations were added to 290 μL of mixed serum from healthy individuals. After sample pretreatment, UPLC-QTOF-MS / MS was performed for detection. A standard curve was plotted with the concentration of each bile acid on the x-axis and (peak area of ​​spiked bile acid / peak area of ​​internal standard - peak area of ​​unspecified bile acid / peak area of ​​internal standard) on the y-axis. The 25 bile acids in the sample were accurately quantified, and the results were expressed as concentration (μmol / L).

[0079] (2) Bile acid metabolism profile analysis

[0080] This invention uses SIMCA-P 14.0 (Umetrics, Sweden) to perform PLS-DA analysis on the data. A discriminant classification model is established using PLS-DA as follows: Figure 2 As shown, the results indicate that the two groups of samples cluster well, exhibiting a clear distinguishing trend, suggesting significant differences in their serum bile acid metabolism profiles. The variable parameter R of this model... 2 Y = 0.638, Q 2 =0.576, indicating that it has good explanatory and predictive capabilities for the variables. The model's prediction accuracy is 91.06%. PLS-DA is a supervised analysis method. To obtain a reliable model and test for overfitting, the model was subjected to 200 permutation tests, and the results are as follows. Figure 3 As shown in the figure, the model does not overfit, indicating that the established model is real and reliable, and there are significant differences in the metabolic profiles of the ICP group and its control group.

[0081] Bile acids with VIP > 1 were selected as candidate biomarkers for ICP. A total of 10 bile acids were screened as potential biomarkers for the diagnosis of ICP, namely T-ω-MCA, TCDCA, GHCA, THCA, TCA, Ta-MCA, GCDCA, GCA, TDCA, and DCA.

[0082] (3) Binary Logistic Regression Analysis and Receiver Operating Curve Analysis

[0083] Using SPSS 25.0 (IBM, USA), binary logistic regression analysis and stepwise regression analysis were performed on 10 potential biomarkers to obtain the combined biomarker T-ω-MCA+GCDCA (P < 0.05). The serum concentration Y of this biomarker was calculated as: Y = 33.152x1 + 2.637x2, where x1 is the concentration of T-ω-MCA and x2 is the concentration of GCDCA. ROC curves were further used to evaluate the diagnostic performance of each single biomarker and the combined biomarker, and the area under the ROC curve (AUC) of each potential biomarker was calculated to determine its diagnostic value. Based on the ROC curves, the threshold value was selected based on the point that best balances sensitivity and specificity, enabling the diagnosis of intrahepatic cholestasis of pregnancy. The AUC, cut-off (μmol / L), sensitivity, specificity, and Youden index of each biomarker in the training set are shown in Table 4. The ROC curves for the combined biomarker T-ω-MCA+GCDCA and the biomarker THCA are shown in [reference needed]. Figure 4 .

[0084] Table 4: ROC curve analysis of serum bile acids in pregnant women with ICP and healthy control pregnant women

[0085]

[0086] Example 2: Clinical sample testing and biomarker effectiveness verification

[0087] This embodiment collected samples from 110 pregnant women, including 29 healthy pregnant women as a control group and 81 women in the ICP group. The ICP group included 12 women (Group A) who presented with abnormal liver function in the mid-to-late stages of pregnancy, but whose total bile acid levels were not elevated at the initial diagnosis. These women developed significant itching or jaundice symptoms 2-4 weeks after the initial diagnosis, and their total bile acid (TBA) levels were higher than normal. Based on their clinical presentation, they were diagnosed with ICP. The other group consisted of 15 women (Group B) with elevated TBA levels at the initial diagnosis and symptoms of itching or jaundice. Both Groups A and B were newly diagnosed patients. ICP is one of the most serious diseases leading to adverse fetal outcomes. Therefore, early detection of ICP patients is a crucial clinical issue. In Group A, patients only presented with abnormal liver function at the initial diagnosis. However, this abnormality was insufficient to differentiate them from other liver-related diseases such as hepatitis. Based on the initial diagnosis, it was impossible to determine whether Group A patients were truly ICP patients, thus delaying timely treatment. The diagnostic criteria followed the "Guidelines for the Diagnosis and Treatment of Intrahepatic Cholestasis of Pregnancy (2015)" issued by the Obstetrics and Gynecology Branch of the Chinese Medical Association (TBA above normal value + comprehensive clinical manifestations).

[0088] Fifty-four patients with intrahepatic cholestasis of pregnancy (ICP) who received drug treatment were designated as group C. In this group, even if TBA levels temporarily returned to normal after treatment, they remained above normal after a period of drug discontinuation. Furthermore, the patients' clinical symptoms (e.g., pruritus and jaundice) persisted, indicating that the ICP was not truly cured. Therefore, these patients should not be considered normal and require continued treatment; they were still diagnosed as ICP patients. This study sampled and tested ICP patients who were not cured after medication and used candidate biomarkers from this protocol to determine whether the patients still had ICP and whether they were cured. The results were compared with the clinical assessments to determine the diagnostic accuracy. The diagnostic criteria followed the "Guidelines for the Diagnosis and Treatment of Intrahepatic Cholestasis of Pregnancy (2015)" published by the Obstetrics and Gynecology Branch of the Chinese Medical Association (TBA above normal + comprehensive clinical manifestations). Generally, drug treatment is given after a diagnosis of ICP. The routine drug treatment regimen is: ursodeoxycholic acid, 250 mg, orally, 2-4 times / day; S-adenosylmethionine, 500 mg, orally, 1-3 times / day. Liver function tests were performed weekly, and the dosage was adjusted based on the clinical manifestations and liver function indicators. The treatment duration was 3 weeks or until delivery. In this technical protocol, group C included 54 ICP patients who had received the above-mentioned conventional treatment regimen. However, after treatment with UDCA, drug-induced UDCA was present in the blood. Since total bile acids, as measured clinically by enzymatic methods, include UDCA, the measurement of total bile acids cannot reflect the true state of the disease. Therefore, it is necessary to find suitable biomarkers that can accurately reflect the patient's condition, given that medication alters the overall metabolite levels and distribution.

[0089] The diagnostic efficacy of each biomarker and TBA in each subgroup was evaluated. Table 5 shows the diagnostic efficacy for groups A and B (disease groups) and healthy pregnant women (control group), and Table 6 shows the diagnostic accuracy in group C. The sample pretreatment and chromatographic / mass spectrometry conditions in this embodiment were the same as in Example 1. The concentrations of each biomarker in the blood sample were detected. Based on the cut-off values ​​in Example 1, predicted positive (true positive + false positive) and predicted negative (true negative + false negative) results were obtained. The detection efficacy of the biomarkers was then determined by comparing the actual positive and actual negative results. Sensitivity, specificity, and diagnostic accuracy were calculated as follows: Sensitivity = TP / (TP+FN) × 100%, which is the percentage of patients who were actually diagnosed with the disease but were correctly identified as having the disease according to the test criteria; Specificity = TN / (FP+TN) × 100%, which is the percentage of patients who were actually not diagnosed with the disease but were correctly identified as not having the disease according to the test criteria; Diagnostic accuracy = (TP+TN) / (TP+FP+FN+TN) × 100%, where TP is the number of true positives, FP is the number of false positives, FN is the number of false negatives, and TN is the number of true negatives. The diagnostic accuracy rate for patients in group C receiving drug treatment was calculated as follows: Diagnostic accuracy rate = Diagnosed as not cured / (Diagnosed as not cured + Diagnosed as cured) × 100%, which is the percentage of patients who were actually not cured but were correctly identified as not cured according to the test criteria. In this study, the total number of patients diagnosed as not cured and those diagnosed as cured was the total number of patients in group C, 54 cases. The blood concentrations of candidate biomarkers in Table 5 were measured in group C. If the concentrations were greater than the threshold (see Table 4 for details), the individual was diagnosed with ICP and counted as the number of people diagnosed as not cured.

[0090] Table 5: Diagnostic efficacy of bile acid markers and TBA in ICP screening (groups A and B as disease groups, healthy pregnant women as control group)

[0091]

[0092] As shown in Table 5, the combined biomarkers T-ω-MCA+GCDCA exhibit high sensitivity, specificity, and diagnostic accuracy in newly diagnosed ICP patients (groups A and B), and can be used for ICP screening and diagnosis, addressing the issue that TBA cannot diagnose early-stage ICP. THCA, with high specificity, can also aid in diagnosis, excluding cases where patients are actually asymptomatic but are diagnosed with the disease (to rule out false positives). Compared to existing technologies, the biomarkers screened in this approach can be used for the prediction and diagnosis of early-stage ICP patients before TBA levels rise, demonstrating a certain predictive power, which is something that existing literature has not reported. In this approach, early-stage ICP patients are defined as those whose TBA levels are not yet elevated at their first visit, but who develop ICP symptoms after a period of disease progression, meeting the diagnostic criteria for ICP. Treatment of these patients in the early stages, before the appearance of obvious symptoms, can effectively improve their pregnancy outcomes and quality of life.

[0093] In the initial screening, 10 bile acids were selected as potential biomarkers for ICP diagnosis. However, when these 10 biomarkers were used in validation samples from initial visits (groups A and B), the biomarkers that showed high VIP and AUC values ​​during screening were not ideal in terms of sensitivity, specificity, and diagnostic accuracy, such as T-ω-MCA and GCDCA. However, combining T-ω-MCA and GCDCA, and calculating the serum concentration of the combined biomarker according to 33.152x1 + 2.637x2, significantly improved sensitivity, specificity, and diagnostic accuracy, achieving unexpected results. Furthermore, TCA and GCA are diagnostic biomarkers reported in existing technologies; however, existing technologies lack validation processes for these two biomarkers, failing to validate their efficacy in initial visits or patients taking medication. This technical approach, for the first time, placed these two candidate biomarkers in validation samples from initial visits for efficacy evaluation, but found that their sensitivity, specificity, and diagnostic accuracy were not ideal. Furthermore, TCA and GCA do not offer any advantage in specificity compared to the combined biomarker T-ω-MCA+GCDCA, nor can they be used to supplement or compensate for the slight deficiency in specificity of the combined biomarker T-ω-MCA+GCDCA. This study overcomes the conventional understanding of existing techniques and identifies new biomarkers with higher accuracy.

[0094] Table 6: Diagnostic accuracy of bile acid markers and TBA in group C

[0095]

[0096] In some ICP patients undergoing drug treatment, total body absorptiometry (TBA) fluctuates with the progress of pregnancy, sometimes temporarily showing normal TBA levels without a cure, which can mislead obstetricians into making incorrect judgments about the condition. In the inventors' prior paper (Cui Yue, Xu Biao, Zhang Xiaoqing et al. Diagnostic and therapeutic profiles of serum bileacids in women with intrahepatic cholestasis of pregnancy-a pseudo-targeted metabolics study.[J]. Clin Chim Acta, 2018, 483:135-141.), a follow-up observation of 11 pregnant women with ICP after medication was reported. The specific treatment was ursodeoxycholic acid (Ursofalk capsules, 250mg, tid). The observation revealed that during ursodeoxycholic acid treatment, the overall trend was a gradual reduction in itching and a significant improvement in quality of life; most ICP patients showed improvement. However, six women had TBA levels below 10 μmol / L, but their liver function and bile acid metabolism had not yet returned to normal levels. This indicates that TBA cannot accurately reflect whether a patient has truly received treatment through medication, necessitating the search for other biomarkers besides TBA capable of monitoring and following up on patient conditions. However, existing technologies, including the inventor's prior research, have not proposed solutions to these problems, nor have they identified biomarkers suitable for disease monitoring and follow-up. Data in Table 6 shows that GCDCA, GCA, and THCA are superior options for disease monitoring and follow-up. The inventor confirmed this in 54 patients with ICP treated with medication, resulting in a larger sample size and more accurate results. Specifically, the diagnostic accuracy of GCDCA reached 0.963, and the diagnostic accuracy of GCA reached 0.944, which was unexpected by those skilled in the art before the experiment.

[0097] Although GCA has been previously reported for ICP diagnosis, its diagnostic accuracy in groups A and B of this invention is not superior to that of the combined diagnostic biomarker of T-ω-MCA and GCDCA, or the biomarker THCA. During follow-up and disease monitoring, medication significantly affects the levels of human metabolites, but GCA is less affected, achieving a diagnostic accuracy of 0.944 in group C, and its serum concentration accurately reflects the patient's condition. Furthermore, the T-ω-MCA+GCDCA combination biomarker exhibited the highest AUC value in the screening of Example 1, leading the inventors to expect it to achieve ideal diagnostic efficacy in diagnosis or disease detection. However, in reality, the T-ω-MCA+GCDCA combination biomarker only showed the most ideal diagnostic efficacy for newly diagnosed patients (groups A and B). When applied to disease monitoring of patients taking medication, its diagnostic accuracy was much lower than that of GCDCA. This indicates that medication and non-medication have a significant impact on the composition and content of total bile acids, and only specific bile acid biomarkers can effectively monitor the disease progression of patients taking medication.

[0098] In summary, TBA is currently the most useful laboratory evidence for diagnosing ICP. However, TBA has low sensitivity for ICP diagnosis and screening, only 0.519, far lower than the combined bile acid markers T-ω-MCA+GCDCA, leading to a high false-negative rate and delaying treatment. For ICP patients receiving drug treatment, the diagnostic accuracy of GCDCA, GCA, and THCA is higher than that of TBA.

[0099] For diagnosis, the following biomarkers can be used: combined biomarker T-ω-MCA+GCDCA, THCA for auxiliary diagnosis; for follow-up, the following biomarkers can be used: GCDCA, GCA, THCA. The specific information of the above biomarkers or combined biomarkers is as follows: Taurine-ω-mouse cholic acid (T-ω-MCA), structural formula see formula (1); Glycinechenodeoxycholic acid (GCDCA), structural formula see formula (2); Taurine-pork cholic acid (THCA), structural formula see formula (3); Glycinecholic acid (GCA), structural formula see formula (4).

[0100]

[0101]

[0102] Comparative Example 1

[0103] Prior to this patent, the inventors had also conducted extensive research on biomarkers for diagnosing ICP. One such study can be found in the inventors' prior paper: "Cui Yue, Xu Biao, Zhang Xiaoqing et al. Diagnostic and therapeutic profiles of serum bile acids in women with intrahepatic cholestasis of pregnancy-a pseudo-targeted metabolomics study.[J]. Clin ChimActa, 2018, 483:135-141." The main problem with the prior research was:

[0104] (1) The sample size was small, with only 55 healthy individuals and 42 patients included.

[0105] (2) Metabolomics analysis was performed only on samples collected at the initial visit, and the conclusions (candidate biomarkers obtained) were not analyzed in samples used for validation, so the efficacy of these biomarkers is still unclear.

[0106] (3) Due to parameter settings issues in UPLC-Triple TOF-MS / MS, some isomers in the sample were not separated, thus failing to identify bile acid biomarkers that more accurately reflect ICP. Specifically, in this study, the single metabolite with the highest AUC value was Gtri-8 (glycine-bound trihydroxy bile acid-8), a substance with an unknown structure. The study only showed that the m / z of the parent ion of Gtri-8 was 464.3018, and its retention time was 11.46 min. Because of its unknown structure, a standard for Gtri-8 could not be found, making accurate quantification and detection impossible in actual testing, thus hindering clinical application. Previous studies concluded that TCA / α-MCA / Gtri-8, as a combined biomarker, are most effective for the diagnosis of ICP. However, due to the aforementioned reasons, this technical approach is difficult to implement in practice.

[0107] The improvement of this technical solution compared to Comparative Example 1 (1):

[0108] In this technical solution, the inventors overcame the aforementioned problems. Regarding sample size, not only samples for biomarker screening but also samples for biomarker validation were included, totaling 179 (91+88) + 110 (29+81) individuals. The increased sample size of 179 (91+88) samples for biomarker screening resulted in more comprehensive screening results. Furthermore, through the validation process, the inventors discovered that not all candidate biomarkers with high AUC values ​​can be used for actual diagnosis. For example, regarding initial diagnostic biomarkers, the screening process in this technical solution found that GCDCA had a relatively ideal AUC value (0.934), and the inventors also indicated in a previous study (Comparative Example 1) that GCDCA's AUC value was relatively ideal (0.917). However, in the subsequent validation process of this solution, it was found that its sensitivity, specificity, and diagnostic accuracy were all unsatisfactory for initial diagnosis, making it unsuitable for initial diagnosis. However, GCDCA can accurately reflect the ICP status of patients receiving drug treatment for follow-up and disease monitoring. Similarly, GCA exhibits the same phenomenon. Although GCA is an ICP biomarker reported in existing technologies, including Comparative Example 1, subsequent validation revealed that its efficacy as a biomarker for early diagnosis and initial diagnosis of ICP is not ideal. However, it showed better results in monitoring the condition of patients already taking medication, making it suitable as a monitoring biomarker rather than an initial diagnostic biomarker for untreated patients. This conclusion demonstrates an unexpected technological advantage achieved through analysis of aspects that existing technologies could not predict.

[0109] Besides GCDCA and GCA, Comparative Example 1 also reported that T-ω-MCA, TCDCA, and TCA had VIP values ​​>1 and could be considered as potential candidate biomarkers. However, Comparative Example 1 only screened the biomarkers in a small sample and did not verify their efficacy. In this technical solution, the inventors tested T-ω-MCA, TCDCA, and TCA in newly diagnosed patients (groups A and B) and patients taking medication (group C). They found that the efficacy of these three candidate biomarkers was unsatisfactory in both cases and unsuitable for use as disease monitoring and diagnostic biomarkers. In particular, T-ω-MCA showed the highest VIP value in the biomarker screening process of this solution (Example 1). However, the diagnostic efficacy of T-ω-MCA was relatively low for both newly diagnosed and medication-taking patients, especially for medication-taking patients, where the diagnostic accuracy was as low as 0.074, which was unexpected by those skilled in the art.

[0110] This technical solution is an improvement (2) compared to Comparative Example 1:

[0111] In addition to the improvements mentioned above, the inventors also adjusted the chromatographic conditions. In their prior research, the chromatographic column used was a Kinetex. A C18 column (50 mm × 2.1 mm, 1.7 μm; Phenomenex) and guard columns C18 ultra-cartridges (2 mm × 2.1 mm) were used. The elution conditions were: mobile phase B 10 min 15%-35%, mobile phase B 5 min increased to 60%, mobile phase B 2 min maintained at 60%, mobile phase B 0.1 min decreased to 15%, and the flow rate was 0.2 ml / min. Compared to Comparative Example 1, the inventors adjusted the above conditions, as detailed in Example 1. Due to the adjustment of the chromatographic conditions, the inventors separated more candidate biomarkers (see...). Figure 1 This approach more accurately reflects the levels of these candidate biomarkers in serum samples, paving the way for a more comprehensive screening of ICP biomarkers. For example, the method in Example 1 did not accurately reflect the serum THCA levels, resulting in unsatisfactory VIP and AUC values ​​for THCA, which prevented it from being included as a candidate biomarker. However, this technical solution improved the sample size and chromatographic conditions, revealing THCA's potential as a primary diagnostic biomarker (due to its high specificity, it can be used to exclude false positives) and its potential as a monitoring biomarker.

[0112] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. The application of biomarkers in the preparation of a system for the diagnosis of intrahepatic cholestasis of pregnancy, characterized in that, The biomarkers include diagnostic biomarkers for diagnosis and screening, which are used for the early diagnosis of intrahepatic cholestasis of pregnancy; the diagnostic biomarkers include a combined diagnostic biomarker consisting of taurine-ω-mouse cholic acid and glycochenodeoxycholic acid.

2. The application of the biomarker according to claim 1 in the preparation of a system for the diagnosis of intrahepatic cholestasis of pregnancy, characterized in that, The serum concentration of the combined diagnostic marker is calculated as follows: 33.152x1 + 2.637x2; where x1 is the serum concentration of taurine-ω-mouse cholic acid and x2 is the serum concentration of glycochenodeoxycholic acid; the diagnostic threshold for the combined marker is 4.41 μmol / L.

3. The application of the biomarker according to claim 1 in the preparation of a system for the diagnosis of intrahepatic cholestasis of pregnancy, characterized in that, The diagnostic markers also include taurine.

4. The application of the biomarker according to claim 3 in the preparation of a system for the diagnosis of intrahepatic cholestasis of pregnancy, characterized in that, The diagnostic threshold for taurine bile acid is 0.0153 μmol / L.

5. A biomarker detection system for the diagnosis of intrahepatic cholestasis of pregnancy, characterized in that, It is used to detect serum concentrations of biomarkers; the biomarkers include diagnostic biomarkers for diagnosis and screening; the diagnostic biomarkers are used for early diagnosis of intrahepatic cholestasis of pregnancy; the diagnostic biomarkers include a combined diagnostic biomarker composed of taurine-ω-mouse cholic acid and glycochenodeoxycholic acid and taurine porcine cholic acid, or the diagnostic biomarker is a combined diagnostic biomarker composed of taurine-ω-mouse cholic acid and glycochenodeoxycholic acid; The marker detection system includes a pretreatment unit, a liquid chromatography unit, and a mass spectrometry unit.

6. The biomarker detection system for the diagnosis of intrahepatic cholestasis of pregnancy according to claim 5, characterized in that, The parameters of the liquid chromatography unit are set as follows: Chromatographic column: Kinetex XB-C18 column, 100 mm in length, 2.1 mm in diameter, and 2.6 μm particle size of packing material; Column temperature: 30℃; Mobile phase: Mobile phase A is 15 mmol / L ammonium acetate solution, and mobile phase B is acetonitrile; Elution method: gradient elution as set in the program below; Flow rate: 0.4 mL / min; Injection volume: 5 μL.

7. The biomarker detection system for the diagnosis of intrahepatic cholestasis of pregnancy according to claim 5, characterized in that, The parameters of the mass spectrometry unit are set as follows: Ion source: electrospray ion source; Mass spectrometry parameter settings: Ion spray voltage: -4500V; Nebulizing gas: 55mL / min; Auxiliary heating gas: 55mL / min; Curtain gas: 25mL / min; Ion source temperature: 550℃; The negative ion scanning mode was used to perform TOF MS first-order mass spectrometry analysis in the range of m / z 70-1000, with a scan time of 500 ms; the precise mass number of each parent ion m / z ± 0.05 Da was used as the target ion, and the daughter ion was scanned in the range of m / z 70-600, with a scan time of 100 ms. For taurine-ω-mouse cholic acid and taurine-pork cholic acid, the parent ion m / z is 514.28, and the daughter ion m / z includes 79.95, 106.98, and 124.00, with a declustering voltage of -60V and a collision energy of -80V. For glycochenodeoxycholic acid, the parent ion m / z is 448.30, the daughter ion m / z is 74.02, the declustering voltage is -60V, and the collision energy is -50V. For glycocholic acid, the parent ion m / z is 464.30, the daughter ion m / z is 74.02, the declustering voltage is -50V, and the collision energy is -50V.

Images