Application of saliva copper ion content detection in hepatolenticular degeneration diagnosis

Abstract

The invention discloses application of saliva copper ion content detection in hepatolenticular degeneration diagnosis, and relates to the technical field of hepatolenticular degeneration disease diagnosis. Saliva specimens of hepatolenticular degeneration patients and saliva specimens of healthy control groups are collected. And comparing the difference of the copper ion levels in saliva of the hepatolenticular degeneration group and the healthy control group by using two independent sample t tests. And analyzing and evaluating the diagnosis performance of the saliva copper ion content by using an ROC curve, and determining a critical value of diagnosis. The result shows that the average saliva copper ion content level of a hepatolenticular degeneration group is 27.0518 [mu] g / L, and the average saliva copper ion content level of a control group is 18.4097 [mu] g / L. The saliva copper ion content of a hepatolenticular degeneration patient is obviously higher than that of a control group (plt; 0.0001) of the method. The area under the curve of the content of the saliva copper ions is 0.8170, which indicates that the content of the saliva copper ions is a good substitute diagnostic marker for distinguishing hepatolenticular degeneration patients.

Description

Technical Field

[0001] The invention relates to the technical field of disease diagnosis of hepatolenticular degeneration, and in particular to an application of saliva copper ion content detection in diagnosis of hepatolenticular degeneration. Background Art

[0002] Hepatolenticular degeneration is a genetic disease of copper metabolism disorder caused by ATP7B gene mutation, with a global incidence of about 1 in 30,000. Copper ions will be abnormally deposited in the patient's liver, brain and other organs, leading to serious consequences such as cirrhosis and nervous system damage.

[0003] Early diagnosis is crucial to improving the prognosis of hepatolenticular degeneration. Currently, diagnostic methods mainly rely on clinical symptoms and signs, laboratory tests (such as serum copper, 24-hour urine copper, serum ceruloplasmin, serum copper oxidase) and genetic testing, and there is a lack of convenient screening methods.

[0004] Saliva testing, as a non-invasive testing method, is simple and easy to operate. As a body fluid, saliva, like serum, contains various ions, amino acids, proteins, hormones, enzymes, electrolytes and other substances, and has great potential value for diagnosing diseases. As a biological sample, saliva is easy to obtain, collect, non-invasive and easy to screen for diseases.

[0005] Chinese patent application CN 118130812A discloses an application of salivary ceruloplasmin in the diagnosis of hepatolenticular degeneration. It was filed by another research group of the applicant on April 1, 2024. The application specification first discovered that a preparation for detecting the level of ceruloplasmin in saliva can be used to prepare a diagnostic preparation for hepatolenticular degeneration. This research group conducted continuous and in-depth research on the saliva of patients with hepatolenticular degeneration, compared the salivary copper levels of patients with hepatolenticular degeneration and normal controls, and for the first time determined that salivary copper can be used to diagnose hepatolenticular degeneration and determine the critical value for diagnosis. Summary of the invention

[0006] The present invention further studies the diagnostic value carrier of saliva, proposes an application of salivary copper ion content detection in the diagnosis of Wilson's disease, and uses salivary copper ion content as a non-invasive diagnostic method for Wilson's disease for the first time.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The application of saliva copper ion content detection in the diagnosis of hepatolenticular degeneration, and the preparation for detecting the copper ion level in saliva can be used in the preparation of diagnostic preparations for hepatolenticular degeneration.

[0009] The diagnostic value of the saliva detection method depends on the accuracy, sensitivity and specificity of distinguishing the diseased from the normal population, which are all indicators for determining the accuracy of the diagnostic test. To this end, the present invention collects saliva samples from patients with Wilson's disease and saliva samples from the healthy control group. The difference in copper ion levels in the saliva of the Wilson's disease group and the healthy control group was compared using two independent sample t-tests. The ROC curve analysis was used to evaluate the diagnostic performance of the saliva copper ion content and determine the critical value for diagnosis. The results show that:

[0010] The average salivary copper ion content level in the Wilson's disease group was 27.0518 μg / L, and that in the control group was 18.4097 μg / L. The salivary copper ion content in Wilson's disease patients was significantly higher than that in the control group (p<0.0001). The area under the curve of salivary copper ion content was 0.8170, indicating that salivary copper ion content is a good alternative diagnostic marker for distinguishing Wilson's disease patients. The critical value of salivary copper ion content is 19.91 μg / L, with a sensitivity of 93.75% and a specificity of 61.90%. This indicates that people with salivary copper ion levels higher than 19.91 μg / L are more likely to be Wilson's disease patients, while those with salivary copper ion levels lower than 19.91 μg / L are more likely to be normal people.

[0011] Based on the significant difference in salivary copper ion content between patients with hepatolenticular degeneration and a normal control group, the present invention determines that the salivary copper ion content can be used as a non-invasive diagnostic method for hepatolenticular degeneration for diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the ROC analysis curve of salivary copper ion content. DETAILED DESCRIPTION

[0013] The present invention is further described in detail below in conjunction with embodiments and drawings.

[0014] This study randomly collected 64 patients diagnosed with Wilson's disease during hospitalization at the First Affiliated Hospital of Anhui University of Chinese Medicine from August 2024 to November 2024. In addition, the study recruited 42 healthy controls without clinical symptoms of Wilson's disease. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Anhui University of Chinese Medicine. Participants were provided with information about the study, and written informed consent was obtained from the patients. Before collecting saliva, all participants underwent a clinical examination of the oral cavity, and there were no oral problems such as gingival bleeding, periodontitis, and oral ulcers.

[0015] The Western medical diagnostic criteria for Wilson's disease are based on a Leipzig score of 4 or above. Saliva is collected from patients in the morning before brushing their teeth, eating, or drinking. Before formal sampling, patients are required to massage both sides of their cheeks for 1 minute, and the patient's tongue rotates in the mouth to promote saliva discharge. Take out the saliva collector and observe the marked scale until the amount of saliva collected is not less than 2 mL. The saliva sample is stored in a -80°C refrigerator as soon as possible and sent to the laboratory for analysis. The sample is thawed at room temperature and then centrifuged at 3000rpm for 10 minutes to remove contaminants before analysis.

[0016] 1. Analysis of saliva copper ion content:

[0017] 1. Determination principle:

[0018] After the sample is digested with nitric acid, it is measured by inductively coupled plasma mass spectrometry and qualitatively analyzed by the specific amount of the element (mass-to-charge ratio, m / z). The external standard method is used to perform quantitative analysis in which the intensity ratio of the mass spectrum signal of the element to be measured and the mass spectrum signal of the internal standard element is proportional to the concentration of the element to be measured.

[0019] 2. Reagents:

[0020] Nitric acid (super pure), Watsons distilled water, multi-element mixed standard.

[0021] 3. Sample determination:

[0022] Measure 1-5 mL of liquid sample into the microwave digestion inner tank. For samples containing ethanol or carbon dioxide, heat them on a hot plate at low temperature to remove the ethanol or carbon dioxide. Add 5-10 mL of nitric acid, cover and place for 1 hour or overnight, tighten the tank cover, and digest according to the standard operating procedures of the microwave digestion instrument. Take it out after cooling, slowly open the tank cover to exhaust, rinse the inner cover with a small amount of water, place the digestion tank on the acid removal instrument to remove the acid, and adjust the volume after the remaining soybean particle size, and filter the membrane. Use ICP-MS to detect the data.

[0023] 4. Instrument conditions (as shown in Table 1):

[0024] Table 1

[0025]

[0026] 5. Calculation of results:

[0027] Element content (μg / mL) = C × V 定容 ÷ V 取样 (1)

[0028] In formula (1), C: concentration of element in solution, μg / L; V 取样 : Volume measured for microwave digestion, mL; V 定容 : Constant volume, L.

[0029] 2. Statistical analysis:

[0030] Data were recorded in Microsoft Excel spreadsheets, analyzed using SPSS 26.0, and plotted using GraphPad Prism 9.0. Descriptive statistics, such as mean and standard deviation, were used to describe the main variables. Kolmogorov-Smirnov test was performed to assess the distribution of variables in order to use parametric or nonparametric tests.

[0031] Since both the control group and the Wilson's disease group were normally distributed, two independent sample t tests were used. Receiver operating characteristic (ROC) analysis was performed to determine the diagnostic value of salivary copper ion content and correctly distinguish Wilson's disease patients from healthy controls. The overall performance was determined by the total area under the curve (AUC), and the optimal diagnostic cutoff value was selected based on its sensitivity and specificity. The statistical significance level was set at p < 0.05.

[0032] 3. Results:

[0033] 1. The Wilson's disease group included 64 Wilson's disease patients, and the control group included 42 healthy subjects. The demographic data of the patients and controls are shown in Table 2.

[0034] Table 2

[0035]

[0036] 2. The salivary copper ion content levels of the two groups are shown in Table 3. The salivary copper ion content level of the Wilson's disease group was significantly higher than that of the control group (p<0.0001).

[0037] Table 3

[0038]

[0039] 3. To determine the diagnostic utility of salivary copper ion content, the tested group was correctly divided into patients with hepatolenticular degeneration and a control group, and ROC analysis was performed (e.g. Figure 1 The total AUC for salivary copper ion content was 0.8170 (as shown in Table 4). The sensitivity and specificity of salivary copper ion content at different levels were measured, and the cut-off value was estimated to be 19.91 μg / L because this produced the best trade-off with a sensitivity of 93.75% and a specificity of 61.9% (as shown in Table 5).

[0040] Table 4

[0041]

[0042] Statistical significance (p<0.05)

[0043] Table 5

[0044]

[0045] The present invention shows through experimental analysis that the saliva copper ion content has good sensitivity and specificity. Therefore, the preparation capable of determining the copper ion level in saliva can be used to prepare the diagnosis preparation of hepatolenticular degeneration.

[0046] The above contents are merely examples and explanations of the concept of the present invention. The technicians in this technical field may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. Application of saliva copper ion content detection in diagnosis of hepatolenticular degeneration, characterized in that: The preparation for detecting the copper ion level in saliva can be applied to the preparation of diagnostic preparation for Wilson's disease.

2. The use according to claim 1, characterized in that There was a significant difference in the salivary copper ion content between the Wilson's disease group and the normal control group.

3. The use according to claim 1 or 2, characterized in that: The detection cut-off value of the preparation for detecting copper ion levels in saliva was 19.91 μg / L, with a sensitivity of 93.75% and a specificity of 61.9%.

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