Application of ergothioneine as biomarker in preparation of rheumatoid arthritis diagnosis product

Abstract

The invention discloses application of ergothioneine as a biomarker in preparation of rheumatoid arthritis diagnosis products, and belongs to the field of biomedicine. According to the invention, the content of ergothioneine in a sample is detected through high performance liquid chromatography-tandem mass spectrometry, so that diagnosis of rheumatoid arthritis of a patient is realized. Through analysis, the AUC (area under curve) of the ergothioneine in distinguishing the RA group from the OA group is 0.7880, and the AUC in distinguishing the RA group from the HC group is 0.8804, which indicates that the ergothioneine has good diagnostic value. According to the technical scheme, a new biomarker is provided for rheumatoid arthritis, a basis is provided for further examination of clinicians, important scientific research and clinical application values are achieved, and a practical means is provided for diagnosis of RA.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of ergothioneine as a biomarker in the preparation of diagnostic products for rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a common systemic autoimmune disease mainly characterized by chronic inflammation of joint tissues, with the main pathological features being a large number of inflammatory cell infiltrations and excessive hyperplasia of synovial tissues at the diseased joints. RA commonly occurs in women and affects approximately 0.5 - 1% of the population worldwide. In China, the incidence rate of RA is approximately 0.28%. The condition of RA progresses rapidly, and the lesions easily lead to joint function decline. Mild functional loss may occur within 1 year of onset, and clinical symptoms such as joint space narrowing, bone destruction, and cystic changes often occur within 2 years, causing irreversible joint bone damage and ultimately resulting in deformity, severely affecting the quality of life of patients. The pathogenesis of RA is complex, and current research results cannot fully clarify its pathogenesis. Existing drugs can only relieve the disease progression by inhibiting the immune response and cannot completely cure RA. Therefore, early diagnosis and timely intervention and treatment are crucial for the prognosis of RA patients.

[0003] Currently, the clinical diagnosis of RA mainly relies on the clinical symptoms of patients, X-ray manifestations, and classical laboratory indicators. However, the early clinical manifestations of RA are diverse, and there are many deficiencies in traditional laboratory diagnostic indicators in clinical practice, making it extremely easy to miss diagnoses and misdiagnoses, thus causing patients to miss the best treatment opportunity. Currently, the traditional laboratory indicators involved in the diagnosis of RA are mainly autoantibodies. According to the classification criteria of the American College of Rheumatology / European League Against Rheumatism (ACR / EULAR) in 2010, these autoantibodies are mainly rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP). However, in actual clinical applications, RF and anti-CCP have quite limitations. According to the 2010 ACR / EULAR standard, when the serological indicators RF and anti-CCP in RA patients are negative, the lesions need to affect more than 10 joints to be diagnosed as RA. Therefore, finding new diagnostic markers to improve the diagnostic efficacy of anti-CCP and RF is of great significance for RA patients, especially for RA patients with negative serological indicators (RF and anti-CCP).

[0004] In our previous non-targeted metabolomics study, we found that the concentration of ergothioneine was decreased in the serum of RA patients, and it has a good effect in diagnosing RA. Ergothioneine (EGT for short) is a sulfur-containing amino acid with antioxidant effects. It is a natural cytoprotectant and antioxidant in the human body, which can scavenge free radicals in the body and protect cells from oxidative damage. In addition, ergothioneine has anti-inflammatory effects, which can reduce the inflammatory response and the level of inflammatory factors. However, little is known about the role of ergothioneine in the diagnosis of RA. Summary of the Invention

[0005] The present invention provides the application of ergothioneine as a biomarker in the preparation of a diagnostic product for rheumatoid arthritis, and it is used for the early diagnosis of rheumatoid arthritis.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The application of ergothioneine as a biomarker in the preparation of a diagnostic product for rheumatoid arthritis.

[0008] The diagnostic product is used to detect the content of ergothioneine in a sample.

[0009] The diagnostic product detects the content of ergothioneine by high performance liquid chromatography-tandem mass spectrometry.

[0010] The above detection method includes: sample pretreatment, chromatographic detection, and mass spectrometric detection.

[0011] The sample pretreatment method includes: thawing the sample, vortexing for 30 s, adding 95 μL of the sample to 380 μL of the extraction solution, vortexing for 30 s, sonicating in an ice water bath for 15 min, leaving the sample to stand at -40 °C for 1 h, and then centrifuging at 4 °C and 12,000 rpm for 15 min to obtain the supernatant; taking the supernatant for on-machine detection.

[0012] The chromatographic detection conditions are as follows: using a Waters ACQUITY UPLC BEH Amide liquid chromatography column; the column oven temperature is 35 °C, and the sample tray is set at 4 °C; mobile phase A is an aqueous solution containing 10 mM ammonium formate and 0.1% formic acid; mobile phase B is acetonitrile; the flow rate is 0.35 mL / min; the injection volume is 1 μL; the gradient elution program is:

[0013] Time / min Phase A / % Phase B / % 0.00 17.00 83.00 1.00 17.00 83.00 3.00 50.00 50.00 4.00 50.00 50.00 4.50 17.00 83.00 9.00 17.00 83.00 .

[0014] The mass spectrometry detection conditions are as follows: ion source: AJS-ESI; detection mode: multiple reaction monitoring mode MRM; scanning mode: positive ion scanning; capillary voltage: +4000 / -3500 V; nozzle voltage: +500 / -500 V; gas N2 temperature 300 °C, gas N2 flow rate 5 L / min, sheath gas N2 temperature 250 °C, sheath gas flow rate 11 L / min, nebulizer pressure 45 psi.

[0015] The extraction solution is prepared by mixing methanol and acetonitrile in a ratio of 1:1.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides a new method for the diagnosis of rheumatoid arthritis. Description of the Drawings

[0018] Figure 1 It is the extracted ion chromatogram of ergothioneine standard in Example 1.

[0019] Figure 2 It is the total ion chromatogram of the sample in Example 1.

[0020] Figure 3 It is the detection standard curve of ergothioneine in Example 1.

[0021] Figure 4 It is the concentration of ergothioneine in plasma in Example 1 (where HD: healthy control; OA: osteoarthritis patient; RA: rheumatoid arthritis patient).

[0022] Figure 5 It is the ROC curve graph of ergothioneine in the diagnosis of RA in Example 1.

[0023] Figure 6 It is the concentration of ergothioneine in plasma in Example 2, where HD: healthy control; OA: osteoarthritis patient; RA: rheumatoid arthritis patient.

[0024] Figure 7 It is the ROC curve graph of ergothioneine in the diagnosis of RA in Example 3. Detailed Embodiments

[0025] Example 1

[0026] I. Preparation of Standard Solution

[0027] Accurately weigh an appropriate amount of ergothioneine standard (CAS: 497 - 30 - 3) into a 10 mL volumetric flask, and prepare a standard stock solution of 10 mmol / L using 50% acetonitrile aqueous solution. Take an appropriate amount of the standard stock solution into a 10 mL volumetric flask, and sequentially dilute the standard solution to obtain a series of calibration solutions. The concentrations of the gradient dilution solutions are: 40000 nmol / L, 20000 nmol / L, 10000 nmol / L, 5000 nmol / L, 2500 nmol / L, 1250 nmol / L, 625 nmol / L, 312.5 nmol / L, 156.25 nmol / L, 78.13 nmol / L, 39.06 nmol / L, 19.53 nmol / L, 9.77 nmol / L, 4.88 nmol / L, 2.44 nmol / L, 1.22 nmol / L.

[0028] II. Sample Pretreatment

[0029] A total of 135 subjects were recruited in this experiment. Take the whole blood samples of the subjects, centrifuge at 3000 rpm for 5 min, and aspirate the supernatant to obtain plasma samples, which are stored at -20 °C for later use. Among them, there are 45 patients with rheumatoid arthritis (RA), 45 patients with osteoarthritis (OA), and 45 healthy controls (HD). The subject samples were all provided by Shanghai Tongren Hospital.

[0030] 1. Take the plasma samples stored at -20 °C and thaw them in an ice - water bath, then vortex for 30 s to mix evenly;

[0031] 2. Take 95 μL of the sample into an EP tube, add 380 μL of the extraction solution (methanol:acetonitrile volume ratio = 1:1, pre - cooled at -40 °C), and vortex for 30 s to mix evenly;

[0032] 3. Ultrasonic in an ice - water bath for 15 min, let the sample stand at -40 °C for 1 hour, and then centrifuge at 4 °C, 12000 rpm (centrifugal force 13800 (×g), radius 8.6 cm) for 15 min to obtain the supernatant;

[0033] 4. Take 90 μL of the supernatant for on - machine detection.

[0034] III. Sample Detection

[0035] 1. Liquid Chromatography Conditions

[0036] The liquid phase system is an Agilent 1290 Infinity II series (Agilent Technologies) ultra - high performance liquid chromatograph;

[0037] Chromatographic column: Waters ACQUITY UPLC BEH Amide (100×2.1 mm, 1.7 μm, Waters) liquid chromatography column;

[0038] Mobile phase A: 10 mM ammonium formate & 0.1% formic acid aqueous solution;

[0039] Mobile phase B: acetonitrile;

[0040] The temperature of the column oven was 35 °C, the sample tray was set at 4 °C, the injection volume was 1 μL, and the gradient elution program was as shown in Table 1 below:

[0041] Table 1 Mobile phase gradient elution program

[0042] Time / min Phase A / % Phase B / % Flow Rate / (mL / min) 0.00 17.00 83.00 0.350 1.00 17.00 83.00 0.350 3.00 50.00 50.00 0.350 4.00 50.00 50.00 0.350 4.50 17.00 83.00 0.350 9.00 17.00 83.00 0.350

[0043] 2. Mass spectrometry conditions

[0044] In this invention, an Agilent 6460 triple quadrupole mass spectrometer equipped with an AJS-ESI ion source was used for mass spectrometry analysis in the multiple reaction monitoring (MRM) mode.

[0045] Table 2

[0046] Compound Name Prec Ion Prod Ion Polarity Quantifier / Qualifier Ergothioneine 230 186.0 Positive Quantifier Ergothioneine 230 126.7 Positive Qualifier Ergothioneine 230 60.0 Positive Qualifier

[0047] The ion source parameters were as follows: Capillary voltage: +4000 / -3500 V, nozzle voltage: +500 / -500 V, gas (N2) temperature: 300 °C, gas (N2) flow: 5 L / min, sheath gas (N2) temperature: 250 °C, sheath gas flow: 11 L / min, nebulizer pressure: 45 psi.

[0048] IV. Calibration curve

[0049] The calibration solution was analyzed by UHPLC-MRM-MS / MS using the method described above.

[0050] The calibration curve of the standard was obtained through analysis. As Figure 3 shown, y represents the peak area ratio of the target compound to the internal standard, and x represents the concentration of the target compound (nmol / L). Least squares regression analysis was performed. When the weight was set to 1 / x, the recovery rate (accuracy) and correlation coefficient (R2) of the calibration solution were the best. If the recovery rate of a certain calibration concentration exceeded the range of 80-120%, the calibration point of that concentration was excluded.

[0051] V. Method Detection Limit and Quantification Limit

[0052] The calibration solution was successively diluted 2-fold and then analyzed by UHPLC-MRM-MS. The detection limit and quantification limit of the method were calculated based on the signal-to-noise ratio. The lowest limit of detection (LLOD) of the method was defined as the compound concentration corresponding to a signal-to-noise ratio of 3, and the LLOD was 39.06 nmol / L. The lowest limit of quantification (LLOQ) of the method was defined as the compound concentration corresponding to a signal-to-noise ratio of 10, and the LLOQ was 78.13 nmol / L.

[0053] VI. Method Precision and Accuracy

[0054] The precision of the method was evaluated by the relative standard deviation (RSD) of repeated injections of QC samples, and the RSD was 4.8%. The accuracy was evaluated by the spiked recovery rate (Recovery) of QC samples. The percentage value of the measured concentration to the spiked concentration was the spiked recovery rate, and the recovery rate at 2000 nmol / L was 86.2%.

[0055] VII. Detection Results of Target Metabolites in Samples

[0056] The content of ergothioneine in the sample was calculated using the detection results obtained by the above detection method of the present invention and the following formula (1):

[0057]

[0058] where CF is the final measured concentration of the sample (in nmol / L), which is obtained by multiplying the directly measured concentration CC (in nmol / L) by the dilution factor Dil (Dilution Factor); CM is the concentration of the target metabolite in the sample (in nmol / L); VF is the final volume of the sample (in μL); and VS is the volume of the sample taken (in μL).

[0059] VIII. Experimental Results

[0060] 1. The extracted ion chromatograms (EICs) of the standard solution and the sample are as Figure 1 - 2 shown. It can be seen from this that: the analytical method adopted in the present invention enables all target compounds to exhibit symmetric chromatographic peaks, and the chromatographic separation of each target compound is well achieved; there are no obvious differences in the retention time and chromatographic peak shape of the target compounds between the biological samples and the standard solution.

[0061] 2. By observing the standard curve for ergothioneine detection, it can be seen that the lower quantification limit of ergothioneine obtained by this method is 78.13 nmol / L, the upper quantification limit is 20,000 nmol / L, and the R 2 is 0.9984.

[0062] 3. The concentration of ergothioneine in patients with HD (healthy control), OA (osteoarthritis), and RA (rheumatoid arthritis) was detected by the above UHPLC-MRM-MS / MS method, and the detection results are as Figure 4 shown. The results showed that the concentration of ergothioneine was decreased in patients with RA (p < 0.0001).

[0063] According to the above detection method, the present invention additionally selected 45 case samples and 45 control samples each to detect ergothioneine in the samples, and thus drew a receiver operating characteristic (ROC) curve graph and evaluated the sensitivity and specificity of the prediction, and further evaluated the evaluation ability of ergothioneine in the detection samples for rheumatoid arthritis.

[0064] For the results, please refer to Figure 5 , Figure 5 which discloses the ROC graph. According to the ROC graph, it can be seen that the area under the curve (AUC) is 0.82, proving that ergothioneine has good diagnostic value in RA.

[0065] Example 2

[0066] In this experiment, 600 subjects were recruited, including 200 cases each of RA, OA patients, and HC. All RA patients met the classification criteria for rheumatoid arthritis of the American College of Rheumatology / European League Against Rheumatism in 2010, and OA patients met the classification criteria for osteoarthritis of the American College of Rheumatology guidelines in 2018. The demographic situation is shown in Table 3.

[0067] Table 3 Information and clinical characteristics of the research subjects

[0068]

[0069] a is expressed as mean ± standard deviation (SD).

[0070] b is expressed as median (25th to 75th percentile).

[0071] The experimental process of this example was the same as that of Example 1. The concentration of ergothioneine in patients with HD (healthy control), OA (osteoarthritis), and RA (rheumatoid arthritis) was detected by the UHPLC-MRM-MS / MS method, and the detection results are as Figure 6 shown. The results showed that the concentration of ergothioneine was decreased in patients with RA (p < 0.0001).

[0072] Example 3

[0073] To verify the diagnostic value of ergothioneine, cases were continuously collected for further verification. A total of 500 RA patients were included in the study, and all patients met the 2010 American College of Rheumatology / European League Against Rheumatism classification criteria for rheumatoid arthritis. In addition, 500 osteoarthritis patients who met the 2018 American College of Rheumatology guidelines and 500 healthy donors were included as control groups. The demographic situation is shown in Table 4.

[0074] Table 4 Information and clinical characteristics of the study subjects

[0075]

[0076] a is expressed as mean ± standard deviation (SD).

[0077] b is expressed as median (25th to 75th percentile).

[0078] Further ROC curve analysis was performed, and the results showed that the area under the curve (AUC) of ergothioneine in differentiating the RA group from the OA group was 0.7880, while the AUC in differentiating the RA group from the HC group was 0.8804 ( Figure 7 ), indicating its good diagnostic value.

[0079] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of ergothioneine as a biomarker in the preparation of a diagnostic product for rheumatoid arthritis.

2. The application according to claim 1, wherein The diagnostic product is used to detect the content of ergothioneine in a sample.

3. The application according to claim 2, characterized in that The diagnostic product detects the content of ergothioneine by high performance liquid chromatography tandem mass spectrometry.

4. The application according to claim 3, wherein The above detection method includes: sample pretreatment, chromatographic detection, and mass spectrometric detection.

5. The application according to claim 4, wherein The sample pretreatment method includes: thawing the sample, vortexing for 30 s, adding 95 μL of the sample to 380 μL of the extraction solution, vortexing for 30 s, sonicating in an ice water bath for 15 min, allowing the sample to stand at -40 °C for 1 h, and then centrifuging at 4 °C and 12,000 rpm for 15 min to obtain the supernatant; taking the supernatant for on-machine detection.

6. The application according to claim 4, wherein The chromatographic detection conditions are: using a Waters ACQUITY UPLC BEH Amide liquid chromatography column; the column oven temperature is 35 °C, and the sample tray is set at 4 °C; mobile phase A is an aqueous solution containing 10 mM ammonium formate and 0.1% formic acid; mobile phase B is acetonitrile; the flow rate is 0.35 mL / min; the injection volume is 1 μL; the gradient elution program is: 。 7. The application according to claim 4, characterized in that, The mass spectrometric detection conditions are: ion source: AJS-ESI; detection mode: multiple reaction monitoring mode MRM; scanning mode: positive ion scan; capillary voltage: +4000 / -3500 V; nozzle voltage: +500 / -500 V; gas N2 temperature 300 °C, gas N2 flow rate 5 L / min, sheath gas N2 temperature 250 °C, sheath gas flow rate 11 L / min, nebulizer pressure 45 psi.

8. The application according to claim 5, characterized in that, The extraction solution is mixed in a ratio of methanol:acetonitrile = 1:1.

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