Application of oxyadipic acid as marker in preparation of kit for detecting diabetic nephropathy

Abstract

The invention relates to the technical field of biomarkers. The invention provides an application of oxyadipic acid as a marker in preparation of a kit for detecting diabetic nephropathy, and also provides an application of a reagent for detecting oxyadipic acid in preparation of a kit for detecting diabetic nephropathy. The oxyadipic acid is used as the marker to distinguish type 2 diabetic patients from diabetic nephropathy patients, so that a basis is provided for diagnosis of diabetic nephropathy.

Description

Technical Field

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

[0002] At present, among the hundreds of millions of diabetic patients, about 40% suffer from diabetic nephropathy, which has become the primary cause of end-stage renal disease and has brought a huge burden to patients' families and society. However, the current diagnosis of diabetic nephropathy still relies on the increase of microalbuminuria and the decrease of glomerular filtration rate. Microalbuminuria is affected by factors such as diet, exercise, and high filtration state of the kidney, and some patients with diabetic nephropathy have a decrease in microalbuminuria, or even always negative. The glomerular filtration rate is mainly estimated by the formula of serum creatinine, which is also affected by diet, muscle content, age, etc. Once the glomerular filtration rate decreases, it often indicates that the kidney disease is more serious and the opportunity for early intervention has been lost.

[0003] Therefore, the development of new diagnostic markers for diabetic nephropathy has become a clinical problem that needs to be solved urgently. Summary of the invention

[0004] The purpose of the present invention is to provide the use of oxyadipic acid as a marker in the preparation of a kit for detecting diabetic nephropathy.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the use of oxyadipic acid as a marker in preparing a kit for detecting diabetic nephropathy.

[0007] The invention provides application of a reagent for detecting oxyadipic acid in preparing a kit for detecting diabetic nephropathy.

[0008] The invention also provides a kit for detecting diabetic nephropathy.

[0009] Preferably, the kit comprises an oxyadipic acid standard and a methanol solution.

[0010] By adopting the above technical solution, the present invention has the following beneficial effects:

[0011] In the technical solution of the present invention, oxyadipic acid is used as a marker, and experiments show that there is a significant difference in the concentration of oxyadipic acid in the urine of patients with type 2 diabetes and diabetic nephropathy. The ROC curve shows that the AUC of this indicator for patients with type 2 diabetes and diabetic nephropathy is 0.70, and the specificity is 100%. This also further illustrates that the oxyadipic acid can be used as a marker to provide an effective basis for the diagnosis of diabetic nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The ROC curve for distinguishing diabetic nephropathy from type 2 diabetes using oxyadipic acid concentration. DETAILED DESCRIPTION

[0013] The present invention provides the use of oxyadipic acid as a marker in preparing a kit for detecting diabetic nephropathy.

[0014] The invention provides application of a reagent for detecting oxyadipic acid in preparing a kit for detecting diabetic nephropathy.

[0015] The invention also provides a kit for detecting diabetic nephropathy.

[0016] In the present invention, the kit includes an oxyadipic acid standard and a methanol solution. The mass volume concentration of the methanol solution in the present invention is preferably 45-55%, more preferably 48-52%, and even more preferably 50%.

[0017] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0018] Example 1

[0019] (I) Selection of test subjects

[0020] Healthy people and patients diagnosed with type 2 diabetes who visited the Second Affiliated Hospital of Nanjing Medical University from January to December 2020, aged 18-75 years, were selected, including 39 cases of diabetic nephropathy, 40 cases of type 2 diabetes, and 37 healthy controls.

[0021] The diagnosis of type 2 diabetes was based on the criteria established by the American Diabetes Association in 2020.

[0022] Healthy controls were selected from the population visiting the hospital during the same period, and had no history of chronic diseases such as obesity, hypertension, diabetes, kidney disease, or long-term medication.

[0023] Diabetic nephropathy is diagnosed based on the above-mentioned diabetes diagnostic criteria, a clear history of diabetes, a causal relationship between urine protein and renal function changes, 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 checked again within 6 months. The urine albumin-creatinine ratio increased twice out of 3 times and other factors such as infection were ruled out.

[0024] Exclusion criteria: patients who had received renal transplantation or dialysis treatment, renal biopsy showed non-diabetic nephropathy, acute stage of infectious diseases, acute kidney injury, or decompensation of other organ functions. (two)

[0026] The fasting morning urine of the above-mentioned research subjects was collected as samples, centrifuged at 4°C and 3000 rpm for 10 min, and after removing impurities, ultraperformance liquid chromatography-tandem mass spectrometry (ACQUITYUPLC-Xevo TQ-S, Waters Corp, Milford, MA, USA) was used for comprehensive targeted quantitative detection of urinary small molecule metabolites.

[0027] Oxyadipic acid standard was purchased from TRC Chemicals (Toronto, ON, Canada). The experiment was performed on an Eppendorf epMotion workstation (Eppendorf Inc., Humburg, Germany). 25 μL of urine sample was added with methanol solution (including internal standard), vortexed vigorously for 5 minutes, and centrifuged at 4°C, 4000g for 30 minutes; derivatization reagent was added and derivatization was performed at 30°C for 60 minutes; 330 μL of 50% methanol was added, and then centrifuged at 4°C, 4000g for 30 minutes, the supernatant was aspirated and mixed with the internal standard for LC-MS analysis, and the concentration of oxyadipic acid was determined, as shown in Table 1.

[0028] Table 1 Concentration of oxyadipic acid in urine samples of different test subjects

[0029]

[0030]

[0031] QuantiChrom by BioAssay Systems TM Creatinine Assay Kit was used to detect the urinary creatinine content in urine samples of different research subjects. The results are shown in Table 2.

[0032] Determination method: (1) Sample addition and standard: Add 5 μL of urine sample and standard to a 96-well plate respectively; (2) Prepare the reaction solution: Prepare the reaction mixture according to 50 μL of A solution, 50 μL of B solution, and 100 μL of double distilled water per well; (3) Add 200 μL of reaction solution to each well and mix thoroughly. Read the value at a wavelength of 510 nm immediately and record it as OD0; read the value at a wavelength of 510 nm again after 5 minutes and record it as OD5; (4) Calculation: Calculate the urine creatinine concentration according to (sample OD5-sample OD0) / (standard OD5-standard OD0)×50 mg / dl.

[0033] Table 2 Urine creatinine content in urine samples of different test subjects

[0034]

[0035]

[0036] The concentration of oxyadipic acid measured by ultra-high performance liquid chromatography-tandem mass spectrometry was corrected by dividing the concentration of oxyadipic acid measured by ultra-high performance liquid chromatography-tandem mass spectrometry by the corresponding urine creatinine to obtain the corrected concentration of oxyadipic acid. The results are shown in Table 3.

[0037] Table 3 Oxyadipic acid concentration in urine samples of different test subjects after correction

[0038]

[0039]

[0040] Statistical analysis was performed according to Table 3. The Wilcoxon rank sum test was used for comparison between the two groups, and the Kruskal-Wallis test was used for comparison between multiple groups. The difference was statistically significant when P < 0.05. The results showed that there was no significant difference in the urine oxygen adipate concentration between patients with type 2 diabetes and healthy controls, while the urine oxygen adipate concentration of patients with diabetic nephropathy was significantly higher than that of the type 2 diabetes group and the healthy control group. (three)

[0042] Based on the above-mentioned urine oxyadipic acid concentrations in 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.

[0043] according to Figure 1 It can be seen that the AUC for distinguishing type 2 diabetes from diabetic nephropathy using urine oxyadipic acid concentration is 0.70, and the specificity is 100%.

[0044] From the above, it can be seen that the technical solution of the present invention uses oxyadipic acid as a marker to distinguish type 2 diabetes patients and diabetic nephropathy patients according to the concentration of oxyadipic acid in urine, providing an effective basis for the diagnosis of diabetic nephropathy.

[0045] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

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

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

3. A kit for detecting diabetic nephropathy, characterized in that: The kit comprises an oxyadipic acid standard and a methanol solution.

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