Application of ergothioneine as a biomarker in the preparation of diagnostic products for rheumatoid arthritis

Abstract

This invention discloses the application of ergothioneine as a biomarker in the preparation of diagnostic products for rheumatoid arthritis (RA), belonging to the biomedical field. This invention uses high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) to detect the content of ergothioneine in samples, thereby achieving the diagnosis of RA in patients. Analysis showed that the area under the curve (AUC) of ergothioneine in distinguishing between the RA and OA groups was 0.7880, while the AUC in distinguishing between the RA and HC groups was 0.8804, indicating its good diagnostic value. This invention provides a novel biomarker for RA, offering a basis for further examination by clinicians, and has significant scientific research and clinical application value, providing a practical means for diagnosing RA.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to the application of ergothioneine as a biomarker in the preparation of diagnostic products for rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is a common systemic autoimmune disease characterized by chronic inflammation of joint tissues. Its main pathological features include extensive inflammatory cell infiltration and excessive synovial tissue proliferation in the affected joints. RA is more common in women, affecting approximately 0.5% to 1% of the global population. In my country, the incidence of RA is approximately 0.28%. RA progresses rapidly, easily leading to joint dysfunction. Mild functional loss may occur within one year of onset, and within two years, clinical symptoms such as joint space narrowing, bone destruction, and cystic changes often appear, causing irreversible joint and bone damage, ultimately leading to deformities and severely impacting the patient's quality of life. The pathogenesis of RA is complex, and current research cannot fully elucidate its mechanisms. Existing drugs can only alleviate disease progression by suppressing the immune response and cannot completely cure RA. Therefore, early diagnosis and timely intervention are crucial for the prognosis of RA patients.

[0003] Currently, the clinical diagnosis of rheumatoid arthritis (RA) mainly relies on patients' clinical symptoms, X-ray findings, and classic laboratory indicators. However, the early clinical manifestations of RA are quite diverse, and traditional laboratory diagnostic indicators have many shortcomings in clinical practice, easily leading to missed diagnoses and misdiagnoses, thus causing patients to miss the optimal treatment window. Currently, the traditional laboratory indicators involved in RA diagnosis are mainly autoantibodies. According to the 2010 American College of Rheumatology / European League of Rheumatology (ACR / EULAR) classification criteria, these autoantibodies are mainly rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP). However, in actual clinical application, RF and anti-CCP have considerable limitations. According to the 2010 ACR / EULAR criteria, when RA patients are negative for serological indicators such as RF and anti-CCP, the lesions need to affect more than 10 joints to be diagnosed with RA. Therefore, finding new diagnostic biomarkers to improve the diagnostic efficacy of anti-CCP and RF is of great significance for RA patients, especially RA patients who are negative for serotonin markers (RF and anti-CCP).

[0004] Our previous non-targeted metabolomics study found that ergothioneine concentrations were reduced in the serum of RA patients, suggesting a potential diagnostic role for RA. Ergothioneine (EGT) is a sulfur-containing amino acid with antioxidant properties. It is a naturally occurring cell protectant and antioxidant in the human body, capable of scavenging free radicals and protecting cells from oxidative damage. Furthermore, ergothioneine has anti-inflammatory effects, reducing inflammatory responses and lowering the levels of inflammatory factors. However, little is known about the role of ergothioneine in the diagnosis of RA. Summary of the Invention

[0005] This invention provides the application of ergothioneine as a biomarker in the preparation of diagnostic products for rheumatoid arthritis, enabling the early diagnosis of rheumatoid arthritis.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] Application of ergothionein as a biomarker in the preparation of diagnostic products for rheumatoid arthritis.

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

[0009] The diagnostic product was tested for ergothionein content using high performance liquid chromatography-tandem mass spectrometry.

[0010] The above-mentioned detection methods include: sample pretreatment, chromatographic detection, and mass spectrometry detection.

[0011] The sample pretreatment method includes: thawing the sample, vortexing for 30 seconds, adding 380 μL of extraction solution to 95 μL of sample, vortexing for 30 seconds, sonicating in an ice-water bath for 15 minutes, placing the sample at -40℃ for 1 hour, and then centrifuging at 4℃ and 12000 rpm for 15 minutes to obtain the supernatant; taking the supernatant for instrumental analysis.

[0012] The chromatographic conditions were as follows: a Waters ACQUITYUPLC BEHAmide column was used; the column oven temperature was 35℃, and the sample tray temperature was set to 4℃; mobile phase A was an aqueous solution containing 10 mM ammonium formate and 0.1% formic acid; mobile phase B was acetonitrile; the flow rate was 0.35 mL / min; the injection volume was 1 μL; and the gradient elution program was as follows:

[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 were as follows: ion source: AJS-ESI; detection mode: multiple reaction monitoring (MRM); scanning mode: positive ion scan; capillary voltage: +4000 / -3500V; nozzle voltage: +500 / -500V; gas N2 temperature: 300℃; gas N2 flow rate: 5L / min; sheath gas N2 temperature: 250℃; sheath gas flow rate: 11L / min; nebulizer pressure: 45psi.

[0015] The extract 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] This invention provides a new method for the diagnosis of rheumatoid arthritis. Attached Figure Description

[0018] Figure 1 The image shows the extracted ion chromatogram of the ergothioneine standard in Example 1.

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

[0020] Figure 3 This is the standard curve for ergothionein detection in Example 1.

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

[0022] Figure 5 This is the ROC curve of ergothionein in the diagnosis of RA in Example 1.

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

[0024] Figure 7 This is the ROC curve of ergothionein in the diagnosis of RA in Example 3. Detailed Implementation

[0025] Example 1

[0026] I. Preparation of Standard Solutions

[0027] Accurately weigh an appropriate amount of ergothioneine standard (CAS: 497-30-3) into a 10 mL volumetric flask and prepare a 10 mmol / L standard stock solution using 50% acetonitrile aqueous solution. Take the corresponding amount of 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 resulting 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, and 1.22 nmol / L.

[0028] II. Sample Pretreatment

[0029] A total of 135 subjects were recruited for this experiment. Whole blood samples were collected from the subjects, centrifuged at 3000 rpm for 5 min, and the supernatant was collected to obtain plasma samples, which were then refrigerated at -20°C for later use. Among them, there were 45 patients with rheumatoid arthritis (RA), 45 patients with osteoarthritis (OA), and 45 healthy controls (HD). All subject samples were provided by Shanghai Tongren Hospital.

[0030] 1. Thaw the plasma sample stored at -20℃ in an ice-water bath and vortex for 30 seconds to mix.

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

[0032] 3. Sonicate in an ice-water bath for 15 min, let the sample stand at -40℃ for 1 hour, and then centrifuge at 4℃ and 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 and test it on the instrument.

[0034] III. Sample Testing

[0035] 1. Liquid Chromatography Conditions

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

[0037] Chromatographic column: Waters ACQUITY UPLC BEH Amide (100×2.1mm, 1.7μm, 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 column oven temperature was set to 35℃, the sample tray temperature to 4℃, the injection volume to 1μL, and the gradient elution program was as shown in Table 1 below:

[0041] Table 1 Gradient elution program for mobile phase

[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] This invention uses an Agilent 6460 triple quadrupole mass spectrometer equipped with an AJS-ESI ion source to perform mass spectrometry analysis in multiple reaction monitoring (MRM) mode.

[0045] Table 2

[0046] CompoundName PrecIon ProdIon 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 are as follows: Capillary voltage: +4000 / -3500V, Nozzle voltage: +500 / -500V, Gas (N2) temperature: 300℃, Gas (N2) flow: 5L / min, Sheath gas (N2) temperature: 250℃, Sheath gas flow: 11L / min, Nebulizer pressure: 45psi.

[0048] IV. Calibration Curve

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

[0050] The standard calibration curve was obtained through analysis, such as Figure 3 As 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, with a weight of 1 / x, yielding the best accuracy and correlation coefficient (R²) for the calibration solution. If the recovery rate of a certain calibration concentration exceeded the range of 80-120%, that calibration concentration was excluded.

[0051] V. Limit of Detection and Limit of Quantification

[0052] The calibration solutions were repeatedly diluted two-fold before being analyzed by UHPLC-MRM-MS. The limits of detection and quantitation were calculated using the signal-to-noise ratio (SNR). The limit of detection (LLOD) was defined as the compound concentration at an SNR of 3, which was 39.06 nmol / L. The limit of quantitation (LLOQ) was defined as the compound concentration at an SNR of 10, which was 78.13 nmol / L.

[0053] VI. Method Precision and Accuracy

[0054] The precision of the method was assessed by the standard relative deviation (RSD) of repeated QC sample injections, which was 4.8%. Accuracy was assessed by the spike recovery of the QC samples; the recovery was the percentage of the measured concentration to the spiked concentration, and it was 86.2% at 2000 nmol / L.

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

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

[0057]

[0058] Wherein, CF is the final measured concentration of the sample (in nmol / L), which is obtained by directly measuring the concentration CC (in nmol / L) by the instrument and multiplying it by the dilution factor Dil; 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 transferred (in μL).

[0059] VIII. Experimental Results

[0060] 1. Extracted ion chromatograms (EICs) of standard solutions and samples, as shown below. Figure 1-2 As shown, the analytical method employed in this invention results in all target compounds exhibiting symmetrical chromatographic peaks, effectively achieving chromatographic separation of each target compound; there are no significant differences in retention time and peak shape between the target compounds in biological samples and standard solutions.

[0061] 2. Observation of the ergothioneine detection standard curve shows that the lower limit of ergothioneine quantification obtained by this method is 78.13 nmol / L, and the upper limit of quantification is 20000 nmol / L. R 2 It is 0.9984.

[0062] 3. The concentrations of ergothioneine in patients with HD (healthy controls), OA (osteoarthritis), and RA (rheumatoid arthritis) were detected using the above-described UHPLC-MRM-MS / MS method. The results are as follows: Figure 4 As shown, the results indicated that ergothioneine concentrations were decreased in RA patients (p<0.0001).

[0063] Based on the above detection method, this invention further selected 45 case samples and 45 control samples to detect ergothionein in the samples, thereby plotting receiver operating characteristic (ROC) curves and evaluating the sensitivity and specificity of the prediction, and further evaluating the ability of ergothionein in the samples to assess rheumatoid arthritis.

[0064] Please see the results. Figure 5 , Figure 5 The ROC plot was published, and the area under the curve (AUC) was 0.82, demonstrating that ergothioneine has good diagnostic value in RA.

[0065] Example 2

[0066] This study recruited 600 participants, including 200 patients with rheumatoid arthritis (RA), 200 with osteoarthritis (OA), and 200 with osteoarthritis (HC). All RA patients met the 2010 American College of Rheumatology / European League Against Rheumatism (ACR / EARD) classification criteria for rheumatoid arthritis, and OA patients met the 2018 ACR guidelines for osteoarthritis. Demographic information is shown in Table 3.

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

[0068]

[0069] 'a' represents the mean ± standard deviation (SD).

[0070] b represents the median (25th to 75th percentile).

[0071] The experimental procedure in this embodiment is the same as in Example 1. The concentration of ergothioneine in patients with HD (healthy controls), OA (osteoarthritis), and RA (rheumatoid arthritis) was detected by UHPLC-MRM-MS / MS method. The detection results are as follows: Figure 6 As shown, the results indicated that ergothioneine concentrations were decreased in RA patients (p<0.0001).

[0072] Example 3

[0073] To validate the diagnostic value of ergothioneine, further case collection was conducted. A total of 500 RA patients were included in the study, all meeting the 2010 American College of Rheumatology / European League Against Rheumatism (ACR / ELAS) classification criteria for rheumatoid arthritis. In addition, 500 patients with osteoarthritis meeting the 2018 ACR guidelines and 500 healthy donors were included as a control group. Demographic details are shown in Table 4.

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

[0075]

[0076] 'a' represents the mean ± standard deviation (SD).

[0077] b represents the median (25th to 75th percentile).

[0078] Further ROC curve analysis showed that the area under the curve (AUC) for ergothionein in distinguishing between the RA and OA groups was 0.7880, while the AUC in distinguishing between the RA and HC groups was 0.8804. Figure 7 This indicates that it has good diagnostic value.

[0079] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of ergothioneine in plasma as a biomarker in the preparation of diagnostic products for rheumatoid arthritis, characterized in that, The diagnostic product is used to detect the content of ergothioneine in a sample, and the content of ergothioneine is detected by high performance liquid chromatography-tandem mass spectrometry.

2. The application according to claim 1, characterized in that, The detection methods include: sample pretreatment, chromatographic detection, and mass spectrometry detection.

3. The application according to claim 2, characterized in that, The sample pretreatment method includes: thawing the sample, vortexing for 30 seconds, adding 380 μL of extraction solution to 95 μL of sample, vortexing for 30 seconds, sonicating in an ice-water bath for 15 minutes, placing the sample at -40℃ for 1 hour, and then centrifuging at 4℃ and 12000 rpm for 15 minutes to obtain the supernatant; taking the supernatant for instrumental analysis.

4. The application according to claim 2, characterized in that, The chromatographic conditions were as follows: a Waters ACQUITY UPLC BEH Amide column was used; the column oven temperature was 35℃, and the sample tray temperature was set to 4℃; mobile phase A was an aqueous solution containing 10 mM ammonium formate and 0.1% formic acid; mobile phase B was acetonitrile; the flow rate was 0.35 mL / min; the injection volume was 1 μL; and the gradient elution program was as follows: 。 5. The application according to claim 2, characterized in that, The mass spectrometry detection conditions were as follows: ion source: AJS-ESI; detection mode: multiple reaction monitoring (MRM); scanning mode: positive ion scan; capillary voltage: +4000 / -3500V; nozzle voltage: +500 / -500V; gas N2 temperature: 300℃; gas N2 flow rate: 5 L / min; sheath gas N2 temperature: 250℃; sheath gas flow rate: 11 L / min; nebulizer pressure: 45 psi.

6. The application according to claim 3, characterized in that, The extract is prepared by mixing methanol and acetonitrile in a ratio of 1:1.

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