Application of phosphatidylcholine as marker in preparation of kit for detecting diabetic kidney disease

Abstract

The invention relates to the technical field of biomarkers. The invention provides application of phosphatidylcholine as a marker in preparation of a kit for detecting a diabetic kidney disease and application of a reagent for detecting phosphatidylcholine in preparation of the kit for detecting the diabetic kidney disease. Phosphatidylcholine is used as the marker to distinguish pure type 2 diabetes patients from diabetic kidney disease patients, so that a basis is provided for diagnosis of the diabetic kidney disease.

Description

Technical Field

[0001] The present invention relates to the technical field of biomarkers, and in particular to the use of phosphatidylcholine as a marker in the preparation of a kit for detecting diabetic nephropathy. Background Art

[0002] Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease and end-stage renal failure in my country. Currently, clinical screening for diabetic kidney disease generally uses the screening indicators estimated glomerular filtration rate (eGFR) and urine albumin / creatinine ratio (UACR). However, DKD has an insidious onset, and using only the estimated values ​​of eGFR and UACR has limitations in the clinical judgment process. Therefore, new biomarkers are needed for the early identification of DKD patients. Studies have shown that DKD has an imbalance in lipid metabolism homeostasis, and the lipid metabolism profile varies at different stages of DKD. With the rise of metabolomics, low-molecular-weight lipids can be comprehensively and systematically analyzed and identified. Therefore, lipid metabolites are expected to be used in the diagnosis of DKD. Summary of the Invention

[0003] The present invention aims to provide the use of phosphatidylcholine as a marker in the preparation of a kit for detecting diabetic nephropathy.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides the use of phosphatidylcholine as a marker in preparing a kit for detecting diabetic nephropathy.

[0006] The present invention also provides the use of a reagent for detecting phosphatidylcholine in preparing a kit for detecting diabetic nephropathy.

[0007] The present invention also provides a kit for detecting diabetic nephropathy, which comprises a phosphatidylcholine standard and a methanol solution.

[0008] By adopting the above technical solution, the present invention has the following beneficial effects:

[0009] The technical solution of the present invention uses phosphatidylcholine (PC) as a marker. Experiments have shown that there is a significant difference in PC concentration in the urine of patients with type 2 diabetes and those with diabetic nephropathy. The receiver operating characteristic (ROC) curve shows that the AUC for this indicator (PC (16:0e|22:5)) is 0.788 for patients with type 2 diabetes and those with diabetic nephropathy, with a sensitivity of 64.5% and a specificity of 88.2%. This further demonstrates that phosphatidylcholine can be used as a marker to distinguish patients with simple type 2 diabetes from those with diabetic nephropathy, providing a basis for the diagnosis of diabetic nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The ROC curve for differentiating type 2 diabetes from diabetic nephropathy using phosphatidylcholine concentration is shown. DETAILED DESCRIPTION

[0011] The present invention provides the use of phosphatidylcholine as a marker in preparing a kit for detecting diabetic nephropathy.

[0012] The present invention also provides the use of a reagent for detecting phosphatidylcholine in preparing a kit for detecting diabetic nephropathy.

[0013] The present invention also provides a kit for detecting diabetic nephropathy.

[0014] In the present invention, the kit includes a phosphatidylcholine standard and a methanol solution.

[0015] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0016] Example 1

[0017] (1) Selection of experimental subjects

[0018] The research subjects were adult patients diagnosed with type 2 diabetes who visited the Second Affiliated Hospital of Nanjing Medical University since 2020, aged no more than 75 years, including 152 cases of diabetic nephropathy and 152 cases of type 2 diabetes.

[0019] The diagnosis of type 2 diabetes was based on the criteria established by the American Diabetes Association in 2020.

[0020] Diabetic nephropathy is diagnosed based on meeting the above-mentioned diabetes diagnostic criteria, a clear history of diabetes, a causal relationship between changes in urine protein and renal function, and a random urine albumin-to-creatinine ratio ≥ 30 mg / g and / or an estimated glomerular filtration rate < 60 ml·min. -1 (1.73m 2 )-1 The urine albumin / creatinine ratio was repeated within 6 months. Two of the three times, the urine albumin / creatinine ratio was elevated, and other factors such as infection were excluded. In addition, the exclusion criteria included previous kidney transplantation or dialysis, renal biopsy findings of non-diabetic kidney disease, acute infectious disease, acute kidney injury, or other organ decompensation. (two)

[0022] Fasting morning urine samples were collected from all study groups and centrifuged at 3000 rpm for 10 minutes at 4°C to remove impurities. Then, 20 μL of urine sample was mixed with a phosphatidylcholine standard (PC (16:0e|22:5) purchased from TRC (Toronto, ON, Canada)) and centrifuged at 13500 g for 10 minutes at 4°C. Then, 30 μL of the supernatant was added with a derivatization reagent (purchased from Sigma–Aldrich) and reacted at 60°C for 1 hour. Then, 400 μL of 50% methanol was added and the mixture was centrifuged at 4000 g for 30 minutes at 4°C. Finally, 135 μL of the supernatant was collected for ultra-performance liquid chromatography-tandem mass spectrometry to determine the concentration of phosphatidylcholine in urine from all study groups. The results are shown in Tables 1 and 2.

[0023] Table 1 Phosphatidylcholine concentration in urine samples of patients with diabetic nephropathy

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[0026] Table 2 Phosphatidylcholine concentration in urine samples of patients with simple type 2 diabetes

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[0030] QuantiChrom by BioAssay Systems TM Creatinine Assay Kit was used to detect the creatinine content in urine samples from different research subjects.

[0031] Assay method: 5 μL of urine sample and creatinine standard were added to a 96-well plate. A reaction mixture was prepared by adding 50 μL of A solution, 50 μL of B solution, and 100 μL of double-distilled water to each well. 200 μL of the reaction mixture was added to each well and mixed. The sample was immediately read at 510 nm, which was recorded as OD0. Five minutes later, the sample was read again at 510 nm, which was recorded as OD5. The creatinine concentration (mg / dL) in the urine sample was calculated as (sample OD5 - sample OD0) / (standard OD5 - standard OD0) × 50 mg / dL. Results are shown in Tables 3 and 4.

[0032] Table 3 Urine creatinine concentration in urine samples of patients with diabetic kidney disease

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[0036] Table 4 Urine creatinine concentration in urine samples of patients with simple type 2 diabetes

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[0040] The phosphatidylcholine concentration measured by ultra-high performance liquid chromatography-tandem mass spectrometry was calibrated by dividing the phosphatidylcholine concentration measured by ultra-high performance liquid chromatography-tandem mass spectrometry by the corresponding urine creatinine to obtain the corrected phosphatidylcholine concentration in urine (nmol / g). The results are shown in Tables 5 and 6.

[0041] Table 5 Phosphatidylcholine concentrations in corrected urine samples of patients with diabetic nephropathy

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[0044] Table 6 Phosphatidylcholine concentrations in corrected urine samples of patients with simple type 2 diabetes

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[0048] Statistical analysis was performed based on the data in Tables 5 and 6. The Wilcoxon rank sum test was used to compare two groups, and the Kruskal-Wallis test was used to compare multiple groups. Differences were considered statistically significant when P < 0.05. The results showed that the urinary phosphatidylcholine concentration in patients with diabetic nephropathy was significantly higher than that in patients with type 2 diabetes. (three)

[0050] Based on the above-mentioned measurement results of phosphatidylcholine concentration in urine of patients with type 2 diabetes and diabetic nephropathy, binary logistic regression analysis was used for statistical modeling, and the subject operating characteristic (ROC) curve was drawn to evaluate the diagnostic efficacy.

[0051] according to Figure 1 It can be seen that the area under the ROC curve (AUC) for distinguishing type 2 diabetes from diabetic nephropathy using urine phosphatidylcholine concentration is 0.788 (95% CI: 0.736, 0.839), with a sensitivity of 64.5% and a specificity of 88.2%.

[0052] In summary, it can be seen that the technical solution of the present invention uses phosphatidylcholine as a marker to distinguish patients with simple type 2 diabetes from patients with diabetic nephropathy, which provides a basis for the diagnosis of diabetic nephropathy.

[0053] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of phosphatidylcholine as a marker in the preparation of a kit for detecting diabetic nephropathy.

2. Application of a reagent for detecting phosphatidylcholine in the preparation of a kit for detecting diabetic nephropathy.

3. A kit for detecting diabetic nephropathy, characterized in that: The kit includes a phosphatidylcholine standard and a methanol solution.

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