A method for early detection of CKD and IgAN based on blood mass spectrometry analysis

Abstract

The application discloses a CKD and IgAN early detection method based on blood mass spectrum analysis, utilizes liquid chromatography-tandem mass spectrometry technology to detect 24 kinds of compounds of a plasma sample in a targeted manner, utilizes a logic regression to establish an early detection model of CKD and IgAN, and verifies the prediction accuracy of CKD and IgAN in a cohort. Through analysis of plasma samples of healthy controls and CKD patients (including IgAN and non-IgAN patients), it is proved that the melatonin or melatonin+tryptophan level in the plasma is an effective method for detecting early CKD, and the melatonin, tryptophan, indole-3-lactic acid, indole-3-carboxylic acid, kynurenic acid and indole-3-methyl aldehyde are effective methods for detecting early IgAN. The application provides effective blood markers for early detection of CKD and IgAN.

Description

Technical Field

[0001] This invention relates to the field of medical and pharmaceutical technology, specifically to an early detection method for CKD and IgAN based on blood mass spectrometry analysis. Background Technology

[0002] Epidemiological surveys show that the prevalence of chronic kidney disease (CKD) is approximately 14.3% worldwide, and about 10.8% in China. Due to its poor prognosis, long treatment period, high treatment costs, and high mortality rate, CKD has become a global public health problem. The course of CKD is progressive; once it progresses to end-stage renal disease (ESRD), patients require dialysis or kidney transplantation to sustain life, placing a heavy emotional and financial burden on patients' families.

[0003] IgA nephropathy (IgAN) is a chronic kidney disease (CKD) characterized by abnormal deposition of IgA immune complexes in the glomerular mesangium. Since its discovery in 1968, IgAN has become the most common type of glomerulonephritis, accounting for 10-20% of primary glomerulonephritis in the United States, 20-30% in some European countries, and 40-50% in developed Asian countries. It has been reported that 40% of IgAN patients develop end-stage renal disease (ESRD) within 10 to 20 years. Currently, early detection of IgAN heavily relies on invasive renal biopsies, which carry risks such as pain and bleeding.

[0004] Early intervention and accurate detection are crucial for preventing the progression of CKD and IgAN. Indicators such as serum creatinine, urea, urinary leukocytes, urinary nitrite, urinary protein, and urinary ketones can be inconsistent with changes in kidney disease. Insensitive and imprecise clinical indicators are insufficient for accurately assessing the condition of CKD and IgAN, posing challenges to corresponding precision treatments. Finding more reliable and convenient early detection methods is of great significance for the precise prevention and treatment of CKD and IgAN.

[0005] Blood mass spectrometry holds great promise for early disease detection. This technology can be used to detect changes in the concentration of various compounds in the body to identify early disease biomarkers and assess disease progression. Currently, this technology has identified some CKD biomarkers, but such studies have mostly focused on moderate to severe kidney damage. Furthermore, blood biomarkers for early detection of IgAN are rarely reported. Summary of the Invention

[0006] The purpose of this invention is to provide an early detection method for CKD and IgAN based on blood mass spectrometry analysis. This method utilizes blood mass spectrometry analysis to find early detection biomarkers for CKD and IgAN, providing an effective method for early warning of CKD and IgAN.

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

[0008] This invention provides an early detection method for CKD and IgAN based on blood mass spectrometry analysis, comprising the following steps:

[0009] S1. Collect plasma samples from several healthy controls, IgAN and non-IgAN patients;

[0010] S2. 24 compounds in plasma samples were specifically detected using liquid chromatography-tandem mass spectrometry.

[0011] S3. Based on the concentrations of 24 compounds in the exploration cohort, establish an early detection model for CKD and IgAN using logistic regression.

[0012] S4. The early detection model based on the exploration queue was examined to verify the prediction accuracy of CKD and IgAN in the queue.

[0013] Furthermore, the 24 compounds in S2 are indole-3-lactic acid, indole-3-carboxylic acid, kynurenic acid, 3-indoleglyoxylic acid, tryptophan, kynurenine, indole-3-carboxaldehyde, indole-3-propionic acid, 3-hydroxykynurenine, 3-hydroxy-anaminobenzoic acid, quinolinic acid, 5-hydroxytryptophan, pyridinecarboxylic acid, serotonin, 5-hydroxy-3-indoleacetic acid, melatonin, 3-indoleacetic acid, 3-indoleacrylic acid, xanthuric acid, N-(3-indoleacetyl)-DL-aspartic acid, 3-indoleacetyl-alanine, N-formylkynurenine, indolemethylglycoside, and indole-3-pyruvic acid.

[0014] Furthermore, the plasma sample is pretreated before detection: a mixed solution of acetonitrile, methanol and formic acid is added to the plasma sample, along with an internal standard solution. After uniform mixing, the sample is sonicated in an ice bath for 10 minutes, incubated at -80°C for 30 minutes, and then centrifuged to collect the supernatant for centrifugation and drying, ready for liquid chromatography-tandem mass spectrometry analysis.

[0015] Furthermore, the volume ratio of the plasma sample to the mixed solution of acetonitrile, methanol, and formic acid is 1:4, the volume ratio of acetonitrile / methanol / formic acid in the mixed solution of acetonitrile, methanol, and formic acid is 50:50:0.1, and the volume of the internal standard solution is 1 / 20 of the plasma sample volume.

[0016] A biomarker for predicting early CKD, the biomarker being selected from melatonin and tryptophan; a biomarker for predicting early IgAN being selected from melatonin, tryptophan, indole-3-lactic acid, indole-3-carboxylic acid, kynurenic acid, and indole-3-carboxaldehyde.

[0017] The use of the markers in detecting and / or predicting early CKD and IgAN is characterized in that the substances used to detect the markers include substances of volumetric or mass concentration of the markers.

[0018] The use of the described markers in the preparation of products for the detection and / or prediction of early CKD and IgAN.

[0019] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects:

[0020] Analysis of plasma samples from healthy controls and CKD patients (including IgAN and non-IgAN patients) demonstrates that plasma melatonin or melatonin + tryptophan levels are effective methods for detecting early CKD. Melatonin, tryptophan, indole-3-lactic acid, indole-3-carboxylic acid, kynurenic acid, and indole-3-carboxaldehyde are effective methods for detecting early IgAN. This invention provides effective blood biomarkers for the early detection of CKD and IgAN. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0022] Figure 1 The present invention provides receiver operating characteristic (ROC) curves for differentiating CKD patients in an exploratory cohort using melatonin, melatonin + tryptophan, and estimated glomerular filtration rate (eGFR).

[0023] In the figure: MEL: melatonin; Trp: tryptophan; eGFR: estimated glomerular filtration rate. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Example 1

[0030] This invention provides a method for early detection of CKD and IgAN based on blood mass spectrometry analysis, comprising the following steps:

[0031] S1. Recruit 202 healthy controls, 70 IgAN patients (CKD stage I-II), and 46 non-IgAN patients (CKD stage I-II, including primary membranous nephropathy and minimal disease nephropathy). Participants were randomly assigned to an exploratory cohort and a validation cohort. The exploratory cohort included 177 healthy controls, 55 IgAN patients, and 36 non-IgAN patients, while the validation cohort included 25 healthy controls, 15 IgAN patients, and 10 non-IgAN patients.

[0032] S2. Collect fasting venous blood samples in the morning from healthy controls, IgAN and non-IgAN patients;

[0033] S3. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to specifically detect 24 compounds in the participants' plasma samples; the LC-MS / MS conditions were as follows:

[0034] Liquid chromatography: 2.1×100mm InfinityLab Poroshell 120 EC-C18 2.7μm column (Agilent, CA), pre-column 2.1×5mm InfinityLab Poroshell 120 EC-C18 2.7μm column (Agilent, CA); injection volume: 5μL (50% methanol-water, containing 0.1% formic acid); column temperature: 40℃; flow rate: 0.4mL / min; phase A: 0.1% formic acid-water; phase B: 0.1% formic acid-acetonitrile; elution gradient as follows:

[0035]

[0036] Mass spectrometry conditions: Scheduled multiple reaction monitoring mode with alternating positive and negative ionization, where the total positive and negative cycle time is 0.65 s. Positive ionization voltage is 5500 V, negative ionization voltage is -4500 V, positive ionization temperature is 550 °C, and negative ionization temperature is 450 °C; curtain gas, ion source gas 1, and ion source gas 2 are 40, 50, and 50 L / h, respectively.

[0037] The 24 compounds detected included:

[0038]

[0039]

[0040] Plasma sample pretreatment: Add 200 μL of acetonitrile / methanol / formic acid (v / v / v, 50 / 50 / 0.1) to 50 μL of plasma, and add 2.5 μL of internal standard solution. Vigorously mix the mixture using a vortex mixer for 20 seconds, sonicate on ice for 10 minutes, incubate at -80°C for 30 minutes, and then centrifuge at 14000×g for 10 minutes at 4°C. Transfer the supernatant to a new Eppendorf tube, dry using a SpeedVac centrifuge, and redissolve the residue with 50 μL of methanol / water / formic acid (v / v / v, 50 / 50 / 0.1), sonicate in ice water for 10 minutes, and centrifuge at 14000×g for 10 minutes at 4°C. Perform LC-MS / MS analysis on the supernatant.

[0041] S4. Based on the concentrations of 24 compounds in the exploration cohort, logistic regression was used to establish early detection models for CKD (including IgAN and non-IgAN) and IgAN. Studies showed that plasma melatonin or melatonin + tryptophan levels are effective methods for detecting CKD, with the former achieving a 95.6% accuracy rate for early CKD detection and the latter achieving 100% accuracy (Table 1). Figure 1Receiver operating characteristic (ROC) curves were used to differentiate CKD patients in an exploratory cohort based on melatonin, melatonin + tryptophan, and eGFR. Furthermore, plasma levels of melatonin, tryptophan, indole-3-lactic acid, indole-3-carboxylic acid, kynurenic acid, and indole-3-carboxaldehyde are early markers for IgAN. Using melatonin + tryptophan to screen for CKD, followed by the combination of indole-3-lactic acid + indole-3-carboxylic acid, indole-3-lactic acid + kynurenic acid, or indole-3-lactic acid + indole-3-carboxaldehyde + indole-3-carboxylic acid to confirm IgAN, the detection accuracy for IgAN reached over 85% (Table 1).

[0042] Table 1. Detection accuracy of CKD and IgAN in the exploration cohort.

[0043]

[0044]

[0045] a. Melatonin; b. Melatonin + Tryptophan; c. Indole-3-lactic acid; d. Indole-3-lactic acid + Indole-3-carboxylic acid; e. 3-Indolelactic acid + Kynucrylamide; f. Indole-3-lactic acid + Indole-3-carboxaldehyde + Indole-3-carboxylic acid

[0046] S5. Based on the cut-off value of the exploratory cohort, CKD and IgAN patients in the validation cohort were identified, as shown in Table 2. The results showed that plasma melatonin or melatonin + tryptophan levels are effective methods for detecting early CKD. Furthermore, melatonin, tryptophan, indole-3-lactic acid, indole-3-carboxylic acid, kynurenic acid, and indole-3-carboxaldehyde are effective methods for detecting early IgAN. Among these, melatonin achieved a 96% accuracy rate in detecting early CKD, and melatonin + tryptophan achieved a 100% accuracy rate. Using melatonin + tryptophan to screen for CKD, followed by the combination of indole-3-lactic acid + indole-3-carboxylic acid, or the combination of indole-3-lactic acid + kynurenic acid, or the combination of indole-3-lactic acid + indole-3-carboxaldehyde + indole-3-carboxylic acid to diagnose IgAN, the detection accuracy for IgAN reached over 93%.

[0047] Table 2. Identification of CKD and IgAN patients in the validation cohort based on cut-off values ​​of the exploratory cohort.

[0048]

[0049] Finally, it should be noted that:

[0050] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A combination of plasma biomarkers for predicting early CKD and IgAN, characterized in that, The markers are selected from melatonin and tryptophan, as well as combinations thereof, including combinations of indole-3-lactic acid and indole-3-carboxylic acid, or combinations of indole-3-lactic acid and kynurenic acid, or combinations of indole-3-lactic acid, indole-3-carboxaldehyde, and indole-3-carboxylic acid.

2. Use of the plasma biomarker combination for predicting early CKD and IgAN as described in claim 1 in the preparation of products for detecting and / or predicting early CKD and IgAN.

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