Methods for differentiating destructive thyroiditis from other pathological conditions

Abstract

The subject of this invention is to provide a method for identifying destructive thyroiditis. This invention relates to a method for identifying destructive thyroiditis, comprising determining at least one of monoiodotyrosine and diiodotyrosine in a sample.

Description

Technical Field

[0001] This invention relates to a method for identifying destructive thyroiditis based on the amount of monoiodotyrosine (MIT) and / or diiodotyrosine (DIT) present outside the thyroid gland, such as in blood. Background Technology

[0002] The thyroid gland is an endocrine organ that produces thyroid hormones from dietary iodine and secretes them into the bloodstream. Two types of thyroid endocrine cells exist. In the follicular lumen, which constitutes the majority of the thyroid gland, tyrosine residues are iodinated in the presence of hydrogen peroxide. Specifically, during the organification of iodine, iodine binds to the carbons at positions 3 and 5 of the tyrosine residues of thyroglobulin (Tg), generating monoiodotyrosine (MIT) and diiodotyrosine (DIT), precursors to thyroid hormones. MIT and DIT then polymerize via peroxidase to generate triiodothyronine (T3), the precursor to thyroid hormones, while DIT and DIT polymerize to generate thyroxine (T4). Within the follicular cells, iodinated Tg releases T3, T4, MIT, and DIT via proteolytic enzymes. T3 and T4 are then secreted into the bloodstream, where iodide ions are released from MIT and DIT by deiodinases and reused. Most thyroid hormones secreted by the thyroid gland into the blood exist in a state bound to proteins in the blood, while the ones that exert their effects are free T3 (FT3) or free T4 (FT4) that are not bound to proteins. The main functions of thyroid hormones are mostly related to life maintenance, including their effects on the heart and skeletal muscle, metabolic connections, the nervous system, and development.

[0003] Destructive thyroiditis is a disease caused by the destruction of thyroid follicles due to stress from childbirth or surgery, or by certain medications or foods, leading to the leakage of thyroid hormones into the bloodstream. This results in symptoms of thyroid toxicity, such as palpitations, weight loss, and excessive sweating. Destructive thyroiditis includes subacute thyroiditis with pain and painless thyroiditis. Unlike Basell's disease, the excessive thyroid hormone state in destructive thyroiditis is temporary. During the recovery period of the damaged thyroid gland, hormone levels decrease, and hypothyroidism may sometimes occur, but it usually resolves after several months.

[0004] In cases of subacute thyroiditis, especially when the affected area is unilateral and there is mild pain, differentiation from undifferentiated thyroid carcinoma and intracystic hemorrhage becomes important. In cases of painless thyroiditis, differentiation from Basel-Dell's disease becomes crucial. Destructive thyroiditis often resolves spontaneously. In cases of severe symptoms, corticosteroids are sometimes administered in subacute thyroiditis, while beta-blockers are sometimes used in painless thyroiditis. Differentiation from Basel-Dell's disease is particularly important. Giving Basel-Dell's disease medication to patients with painless thyroiditis can easily lead to hypothyroidism, and the side effects of Basel-Dell's medication (especially granulocytopenia) can become a major problem.

[0005] As a method for differentiating between destructive thyroiditis and Basell's disease, a common practice is to further measure anti-TSH receptor antibody (TRAb) when the patient's FT3 and FT4 levels are high and thyroid-stimulating hormone (TSH) is below 0.1 μIU / mL. A positive result indicates Basell's disease, while a negative result indicates destructive thyroiditis. TRAb measurement is performed using a kit, but the antibodies used for TRAb measurement are not limited to a single antibody; for example, antibodies from idiopathic myxedema can also result in high values, thus raising issues with the properties and measurement methods. Furthermore, TRAb can occasionally be positive in painless thyroiditis and subacute thyroiditis (Non-Patent Literature 1-4).

[0006] The following methods are known as differentiating other destructive thyroiditis and Basell's disease.

[0007] (i) FT3 / FT4 ratio

[0008] If the FT3 / FT4 ratio increases to the same extent, it indicates painless thyroiditis. If the FT3 / FT4 ratio is above 2.5, it indicates Basel-Dürer's disease, but the sensitivity and specificity are low.

[0009] (ii) Blood flow index or blood flow density

[0010] In thyroid ultrasound examination, decreased blood flow indicates painless thyroiditis, while increased blood flow indicates Basel disease.

[0011] (iii) Blood flow velocity in the inferior thyroid artery

[0012] If the velocity ranges from normal to low, it indicates painless thyroiditis; if it ranges from normal to abnormally high, it indicates Basel-Dürer's disease. However, there are also cases where low values ​​are observed in Basel-Dürer's disease and high values ​​are observed in painless thyroiditis.

[0013] (iv) 123 I or 99m Tc scintillation scan (uptake rate)

[0014] like 123 I or 99m A decrease in Tc (taken through the same mechanism as iodine) uptake indicates painless thyroiditis, while an increase from normal levels indicates Basel-Dürer's disease. 99m Tc is a time-grade radioactive isotope, and the inspection cannot be cancelled; in many cases, the identification process ends upon completion of the preparation. On the other hand, 99m Tc has the advantage of not having iodine intake restrictions, but it cannot detect large amounts of iodine intake.

[0015] (v) Measurement of urinary iodine excretion

[0016] An increase in total iodine excretion in urine indicates painless thyroiditis, while no increase indicates Basel-Dürer's disease. However, the timing of the test is limited due to the influence of food intake.

[0017] As described above, various methods have been developed to differentiate destructive thyroiditis, but there is no simple and accurate method for differentiation.

[0018] However, as mentioned above, MIT is a precursor of thyroid hormones, consisting of iodine coordinated at the meta position of the phenolic ring of tyrosine. The two molecules combine to form 3,3'-diiodothyronine. Additionally, in the thyroid colloid, another molecule binds to diiodotyrosine to form triiodothyronine.

[0019] Furthermore, as mentioned above, DIT is a precursor to thyroid hormones, consisting of a substance with iodine coordinated at the meta position of the phenolic ring of monoiodotyrosine. Diiodotyrosine regulates the activity of iodide peroxidase. Additionally, it binds to monoiodotyrosine in thyroid colloids to form triiodothyronine. When two molecules of diiodotyrosine combine, thyroxine can be formed.

[0020] The use of MIT and DIT as anticancer agents is known (Patent Document 1), but the release of these substances into the bloodstream after the thyroid gland is destroyed is unknown.

[0021] Existing technical documents

[0022] Patent documents

[0023] Patent Document 1: Japanese Patent Publication No. 7-116031

[0024] Non-patent literature

[0025] Non-patent literature 1: Ikeda Sai, "25. Hyperthyroidism and Hypothyroidism", Clinical Examination Guidelines for Diagnostic Groups 2003 - Standardization for Medical Treatment, Japan Society of Clinical Laboratory Medicine, pp. 107-110, 2003.

[0026] Non-Patent Literature 2: Hiroshi Yoshimura: Research on abnormal thyroid hormones, Journal of the Japanese Society of Internal Medicine, Vol. 103, No. 4 (April 10, 2018): 855-861

[0027] Non-Patent Literature 3: Katsumi Yoshida et al.: Diagnosis of subacute thyroiditis and painless thyroiditis, Journal of the Japanese Society of Internal Medicine, Vol. 86, No. 7 (July 10, 2008): 48-53

[0028] Non-patent literature 4: Ayuko Nakano et al.: A rapid differential diagnosis between Basell's disease and destructive thyroiditis at the initial diagnosis, Dokkyo Journal of Medical Science, 36(2), 105-108, 2009 Summary of the Invention

[0029] The subject of this invention is to provide a method for identifying destructive thyroiditis.

[0030] The inventors of this invention conducted in-depth research to solve the above-mentioned problems and found that, in patients with destructive thyroiditis, compared with patients with thyroid diseases other than destructive thyroiditis and thyroid dysfunction (hereinafter, both are collectively referred to as "non-destructive thyroiditis diseases"), such as patients with TSH and FT4 within the baseline range or patients with Basel-Dürer's disease, the amount of monoiodotyrosine and diiodotyrosine released into the blood after the thyroid gland is unknowingly destroyed is unexpectedly higher, thus completing the present invention.

[0031] That is, the present invention is as follows.

[0032] [1] A method for identifying destructive thyroiditis, comprising determining at least one of monoiodotyrosine and diiodotyrosine in a sample.

[0033] [2] According to the identification method described in the above item [1], the determination of monoiodotyrosine and diiodotyrosine.

[0034] [3] The identification method described in item [1] or [2] above, wherein the sample is blood.

[0035] [4] The identification method according to any one of the above items [1] to [3], wherein at least one of monoiodotyrosine and diiodotyrosine in the sample is determined by LC-MS, and the LC-MS uses their stable isotopes as internal standards.

[0036] [5] A biomarker for identifying destructive thyroiditis, characterized in that the biomarker is either monoiodotyrosine or diiodotyrosine in the sample.

[0037] [6] The biomarkers described in the above item [5] are monoiodotyrosine and diiodotyrosine.

[0038] [7] The biomarkers described in the above items [5] or [6], wherein the sample is blood.

[0039] [8] A kit for identifying destructive thyroiditis, characterized in that the kit comprises: a unit for measuring at least one of monoiodotyrosine and diiodotyrosine in a sample collected from a subject; and a unit for comparing the measured value of the subject with the measured value of at least one of monoiodotyrosine and diiodotyrosine in corresponding samples collected from a non-patient, a patient with non-destructive thyroiditis, and a patient with destructive thyroiditis.

[0040] [9] A kit for identifying destructive thyroiditis, characterized in that the kit comprises: a unit for determining at least one of monoiodotyrosine and diiodotyrosine in a sample collected from a subject; and a unit for comparing the measured value of the subject with the cutoff value of monoiodotyrosine and / or diiodotyrosine.

[0041]

[10] The identification kit according to the above item [8] or [9], wherein monoiodotyrosine and diiodotyrosine are determined.

[0042] According to the present invention, destructive thyroiditis can be identified. In particular, destructive thyroiditis can be differentiated from non-destructive thyroiditis diseases, such as Basel-D, Prometheus, and transient hyperthyroidism of pregnancy, especially Basel-D. Attached Figure Description

[0043] Figure 1 This is a graph showing the concentration of monoiodotyrosine (MIT) in the serum of patients with thyroid disease.

[0044] Figure 2 This is a graph showing the concentration of diiodotyrosine (DIT) in the serum of patients with thyroid disease.

[0045] Figure 3 This is a graph showing the concentration of MIT in the serum of patients with thyroid disease.

[0046] Figure 4 It is represented by quartiles (box plot). Figure 3 The result is shown in the graph.

[0047] Figure 5 This is a graph showing the concentration of DIT in the serum of patients with thyroid disease.

[0048] Figure 6It is represented by quartiles (box plot). Figure 5 The result is shown in the graph.

[0049] Figure 7 It is Figure 1 and Figure 3 The results are summarized in a graph.

[0050] Figure 8 It is represented by quartiles (box plot). Figure 7 The result is shown in the graph.

[0051] Figure 9 It is Figure 2 and Figure 5 The results are summarized in a graph.

[0052] Figure 10 It is represented by quartiles (box plot). Figure 9 The result is shown in the graph.

[0053] Figure 11 This is a graph representing the standard curve of DIT. Detailed Implementation

[0054] In this invention, as previously stated, "non-destructive thyroiditis" refers to thyroid diseases and thyroid dysfunction other than destructive thyroiditis, such as Basell's disease, Prometheus disease, and transient hyperthyroidism during pregnancy. Furthermore, "non-destructive thyroiditis" also includes diseases involving total thyroidectomy with administration of levothyroxine (LT4), diseases with abnormal thyroid test values ​​that are not classified as thyroid diseases, and diseases where thyroid hormone and TSH levels are within the normal range during treatment for thyrotoxicosis (hyperthyroidism) or hypothyroidism.

[0055] In this invention, since the amount of at least one of monoiodotyrosine and diiodotyrosine (hereinafter collectively referred to as "the biomarkers of this invention") in samples collected from patients with destructive thyroiditis, non-patients, and patients with non-destructive thyroiditis is higher in patients with destructive thyroiditis than in non-patients and patients with non-destructive thyroiditis, destructive thyroiditis can be identified by measuring the biomarkers of this invention, and in particular, destructive thyroiditis can be distinguished from non-destructive thyroiditis (especially Basel-Dürer's disease).

[0056] Specifically, for example, by measuring at least one of the biomarkers of the present invention in samples collected from a subject, a non-patient, and a patient with non-destructive thyroiditis, and comparing the measured values ​​of the subject with those of the non-patient and non-destructive thyroiditis patients, it is possible to identify whether the subject has destructive thyroiditis. For example, if, by statistical methods, the measured value of the subject is significantly higher than that of the non-patient and non-destructive thyroiditis patients, the subject can be identified as having destructive thyroiditis rather than non-destructive thyroiditis.

[0057] Alternatively, destructive thyroid disease can be identified by setting a cutoff value and comparing the measured values ​​of the subject with the cutoff value. The cutoff value is the value at which the subject is identified as having destructive thyroiditis rather than non-destructive thyroiditis when the measured values ​​of MIT and / or DIT in the sample exceed the value.

[0058] The cutoff value varies depending on the animal species and the sample. For example, the cutoff value for MIT in human serum is preferably 250 pg / mL (e.g., 260 pg / mL), more preferably 210 pg / mL (e.g., 200 pg / mL), further preferably 160 pg / mL, and particularly preferably 120 pg / mL. Similarly, the cutoff value for DIT in human serum is, for example, 400 pg / mL, preferably 300 pg / mL (especially 350 pg / mL), more preferably 260 pg / mL, further preferably 250 pg / mL, and particularly preferably 200 pg / mL.

[0059] The cutoff value can be calculated, for example, based on the ROC (Receiver Operating Characteristic) curve.

[0060] In this invention, identification can be performed by comparing the determination of at least one of monoiodotyrosine and diiodotyrosine, but it is particularly preferred to determine diiodotyrosine for comparison. Furthermore, in this invention, to improve the precision and accuracy of identification, it is preferable to determine both monoiodotyrosine and diiodotyrosine for comparison.

[0061] In the identification method of this invention, "subject" refers to an animal including humans, "non-affected" refers to an animal including humans whose TSH and FT4 are within the baseline range during treatment for thyroid diseases and thyroid dysfunction such as thyrotoxicosis and hypothyroidism, "non-destructive thyroiditis disease patient" refers to an animal including humans diagnosed by a physician or veterinarian as having non-destructive thyroiditis, and "destructive thyroiditis patient" refers to an animal including humans diagnosed by a physician or veterinarian as having destructive thyroiditis.

[0062] Furthermore, the baseline values ​​for TSH and FT4 mentioned above can be any values ​​that do not show significant abnormalities compared to healthy humans or healthy animals other than humans. These values ​​vary depending on the animal species and the sample. For example, the baseline values ​​for TSH in human serum can be 0.61-4.68 μIU / mL, and the baseline values ​​for FT4 in human serum can be 0.76-1.65 ng / dL.

[0063] Examples of animals other than humans include pets such as dogs, cats, and parrots; livestock such as cows, pigs, and horses; and poultry such as chickens.

[0064] In the identification process of this invention, when comparing the measured values ​​of the test subject, non-affected individuals, individuals with non-destructive thyroiditis, and individuals with destructive thyroiditis, or when comparing the measured values ​​of the test subject with the cutoff value, it is preferable that these values ​​are measured in at least the same animal species. Furthermore, it is preferable that the conditions of subspecies (including human race), sex, age (including months and weeks), height and weight, and the sample are also the same.

[0065] In this invention, the "sample" used to determine MIT and DIT refers to a sample collected from a subject, a non-affected individual, a patient with non-destructive thyroiditis, or a patient with destructive thyroiditis. There are no particular restrictions on the biological source of the sample, as long as it is not from the thyroid gland; any sample can be used. Examples include blood (whole blood, serum, plasma), saliva, tears, sweat, urine, feces, bile, tissue, hair, or biological cells, or cultures of tissues or biological cells, or preparations obtained from organs (wherein, organs do not include the thyroid gland, and tissues and cells do not include thyroid tissue and cells). Blood, saliva, and urine are preferred, with blood, especially serum and plasma, being more preferred.

[0066] In this invention, the method for determining the biomarkers of this invention is not particularly limited as long as it can determine monoiodotyrosine and / or diiodotyrosine in the sample, and conventional determination methods can be used.

[0067] For example, conventional radioimmunoassay (RIA) (Elevated serum diiodotyrosine (DIT) in severe infections and sepsis: DIT, a possible new marker of leukocyte activity. Meinhold H, Gramm HJ, Meissner W, Zimmermann J, Schwander J, Dennhardt R, Voigt KJ Clin Endocrinol Metab. 1991 Apr; 72(4):945-53) or LC-MS / MS (Effects of exogenous monoiodotyrosine on the serum levels of anterior pituitaryhormones. Tan SA, Lewis JE, Berk LS, Wilcox RB. Acta Endocrinol (Copenh). 1991 Mar; 124(3):251-7) can be used. In addition, chemiluminescent enzyme immunoassay (CLEIA), enzyme-linked immunosorbent assay (ELISA), and electrochemiluminescent immunoassay (ECLIA) can also be used.

[0068] Preferably, liquid chromatography-mass spectrometry (LC-MS) with a wide quantitative range is used, which does not require the use of radioactive materials or derivatization, and can use stable isotopes as internal standards (IS).

[0069] LC-MS refers to an analytical method that uses a combination of liquid chromatography and mass spectrometry, such as liquid chromatography-tandem mass spectrometry (LC-MS / MS) and liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS), with LC-MS / MS being the preferred method.

[0070] For quality analysis, the target component needs to be ionized. Ionization methods include atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), and atmospheric pressure photoionization (APPI). These ionization methods are combined with LC-MS / MS, using methods such as Liquid Chromatography-Electrosprayionization based Tamdem Mass Spectrometry (LC-ESI / MS / MS) and Liquid Chromatography-Atmospheric pressure chemical ionization based Tamdem Mass Spectrometry (LC-APCI / MS / MS). These measurements can be performed using general methods. LC-ESI / MS / MS is preferred in this study. Ion detection includes positive and negative ion detection; negative ion detection is preferred in this study.

[0071] In addition, quality analysis units can include magnetic field type, quadruple pole type, time-of-flight type, etc., but in this invention, the quadruple pole type, which has good quantification, wide dynamic range and good linearity, is preferred.

[0072] Furthermore, examples of ion detection in quantitative analysis include selective ion monitoring (SRM) and selected reaction monitoring (SRM). SRM selectively detects only the target ion, while SRM selects one type of ion generated by the first mass analysis unit as a precursor ion, and the second mass analysis unit detects the product ions generated by the splitting of this precursor ion. In this invention, SRM-based determination, which improves the signal-to-noise ratio by increasing selectivity and reducing noise, is preferred.

[0073] Stable isotopes of monoiodotyrosine and diiodotyrosine can be commercially available or synthesized using conventional methods.

[0074] The advantage of the identification method of the present invention is that, since MIT or DIT is the object of measurement, the above-mentioned negative ion detection LC-ESI / MS / MS method can be used to easily and accurately identify destructive thyroiditis.

[0075] The present invention also relates to a method for using monoiodotyrosine or diiodotyrosine, preferably both, as biomarkers for identifying destructive thyroiditis, particularly for differentiating destructive thyroiditis from non-destructive thyroiditis (especially Basel-Dürer's disease).

[0076] The present invention also relates to an identification kit for use in the identification method of the present invention.

[0077] The kit used in the identification method of the present invention may contain at least one selected from buffers, acids, bases, alcohols, syringes, antibodies, and documents describing the measurement procedures. Furthermore, the kit used in the identification method of the present invention may also contain documents describing standards.

[0078] More specifically, the "identification kit" of the present invention includes a unit for identifying destructive thyroiditis by comparing the measured value of at least one of the biomarkers of the present invention in a sample collected from a subject with the measured value or cutoff value of at least one of the biomarkers of the present invention in the corresponding samples collected from non-affected individuals, patients with non-destructive thyroiditis, and patients with destructive thyroiditis. For example, as a unit for measuring at least one of the biomarkers of the present invention present in a sample collected from a subject, examples include sample collection or preparation equipment, measuring equipment or reagents for MIT and / or DIT, such as a column. Furthermore, as a comparison unit for comparing the measured value of at least one of the biomarkers of the present invention in a sample collected from a subject with the measured value or cutoff value of at least one of the biomarkers of the present invention in the corresponding samples collected from non-affected individuals, patients with non-destructive thyroiditis, and patients with destructive thyroiditis, examples include a comparison table of the measured values ​​or cutoff values ​​for each of the three groups. This comparison table is particularly useful because it can identify diseases even without a doctor's judgment.

[0079] The present invention also relates to a method for screening a treatment or preventive agent for destructive thyroiditis, the method comprising determining at least one of monoiodotyrosine and diiodotyrosine in a sample, preferably both.

[0080] Example

[0081] The present invention is described below with reference to specific embodiments, but the present invention is not limited to the embodiments described below. Furthermore, the apparatus and materials used in the embodiments are described below.

[0082] [Example 1] Differential diagnosis of destructive thyroiditis patients based on LC-MS / MS measurements (1)

[0083] 1. Preparation of samples containing MIT and DIT

[0084] Add 0.05 mL of human serum and internal standard (300 pg / 0.05 mL MIT) to the test tube. 13 C6 and DIT- 13 C9 15Add 0.05 mL of a mixed methanol solution of N to 0.1 mL of a 25 mg / mL dithiothreitol / 1% pyridine aqueous solution, stir, and let stand at room temperature for 30 minutes. After standing, add 1 mL of purified water and 4 mL of methyl tert-butyl ether to a test tube, shake for 5 minutes, and centrifuge for 3 minutes to remove methyl tert-butyl ether. Add 0.5 mL of acetic acid / acetonitrile (1:50) to the aqueous layer, centrifuge for 3 minutes, transfer the supernatant to another test tube, and load it onto a CX column (Thermo-Fisher) pre-conditioned with 3 mL of methanol, 1 mL of purified water, and 1 mL of 1% acetic acid aqueous solution. Wash with 1 mL of purified water and 3 mL of methanol, dissolve MIT and DIT in 1 mL of methanol / purified water / ammonia (6:14:1), distill the eluent using a centrifugal evaporator, and dissolve it in 0.1 mL of 20 mM ammonium formate aqueous solution to prepare a sample containing MIT and DIT.

[0085] 2.LC-MS / MS conditions

[0086] The prepared samples were analyzed by LC-MS / MS. The LC-MS / MS conditions are shown in Table 1 below.

[0087] [Table 1]

[0088] LC-MS / MS analysis conditions

[0089]

[0090] Analytical analyte / internal standard

[0091]

[0092] Measurement interval: 1.2–2.5 min

[0093] Resolution: Q1 / Unit Q3 / Unit

[0094] 3. Validation results

[0095] The validity verification results of the above LC-MS / MS analysis are shown in Table 2 below.

[0096] [Table 2]

[0097]

[0098] As shown in Tables 1 and 2, the determination time for each sample of the MIT and DIT based LC-MS / MS method of the present invention is as short as 3 minutes, the limit of quantitation is 60 pg / mL, and the effectiveness verification results are also good.

[0099] The MIT and DIT levels in the serum of patients with thyroid diseases were measured using the LC-MS / MS-based method of the present invention described above. The results are shown in Table 3 and... Figure 1 and 2 .

[0100] [Table 3]

[0101]

[0102] Control: Patients with abnormal physical or data presentations suspected of having thyroid disease, despite TSH / FT4 measurements, whose TSH and FT4 levels remain within the baseline range.

[0103] DT: Patients diagnosed with destructive thyroiditis (DT) (1 subacute, 2 painless).

[0104] GD: Patients diagnosed with Basell's disease (GD)

[0105] GTH: Transient hyperthyroidism during pregnancy

[0106] From Table 3 and Figure 1 and 2 It is known that destructive thyroiditis can be differentiated by measuring MIT or DIT in the blood. In particular, it is known that destructive thyroiditis can be differentiated from non-destructive thyroid diseases, especially Basel-Dürer's disease.

[0107] [Example 2] Differential diagnosis of destructive thyroiditis patients based on LC-MS / MS measurements (2)

[0108] Following the method described in Example 1, MIT and DIT levels in the serum of patients with thyroid disease were determined. The results are shown in Table 4 and... Figure 3-6 .

[0109] [Table 4]

[0110]

[0111] Control: In a physician's diagnosis, there are physical abnormalities or abnormal data indicating suspected thyroid disease, where TSH and FT4 levels remain within the baseline range despite TSH / FT4 testing.

[0112] DT: Patients diagnosed with destructive thyroiditis (DT)

[0113] GD: Patients diagnosed with Basell's disease (GD)

[0114] From Table 4 and Figure 3-6It is known that destructive thyroiditis can be differentiated by measuring MIT or DIT in the blood. In particular, it is known that destructive thyroiditis can be differentiated from Basel-Dürer's disease.

[0115] [Example 3] Differential diagnosis of destructive thyroiditis patients based on LC-MS / MS measurements (3)

[0116] The results from Examples 1 and 2 were summarized and the data were calculated. The results are shown in Table 5 and... Figure 7-10 .

[0117] [Table 5]

[0118]

[0119] Control: In a physician's diagnosis, there are physical abnormalities or abnormal data indicating suspected thyroid disease, where TSH and FT4 levels remain within the baseline range despite TSH / FT4 testing.

[0120] DT: Patients diagnosed with destructive thyroiditis (DT)

[0121] GD: Patients diagnosed with Basell's disease (GD)

[0122] GTH: Transient hyperthyroidism during pregnancy

[0123] Table 5 and Figure 7-10 The results are a summary of the results from Examples 1 and 2, thus it can be seen that with the increase in the number of examples, the accuracy is further improved.

[0124] From Table 5 and Figure 7-10 It is known that destructive thyroiditis can be differentiated by measuring MIT or DIT in the blood. In particular, it is known that destructive thyroiditis can be differentiated from non-destructive thyroid diseases, especially Basel-Dürer's disease.

[0125] [Example 4] Determination of DIT based on chemiluminescent enzyme immunoassay (CLEIA)

[0126] 1. Preparation of plates and solutions

[0127] The preparation of a 96-well plate for immobilized anti-mouse IgG rabbit antibody was as follows: The anti-mouse IgG rabbit antibody solution (Jackson Immunoresearch) was diluted to a concentration of 10 μg / mL with phosphate-buffered saline, and 100 μL of this solution was dispensed into 96-well empty plates (Thermofisher Scientific). After reacting at 4°C for 16 hours, the plate was washed twice with 0.02% Triton X-100 (registered trademark) and then twice with 5% sucrose. The alkaline phosphatase-labeled DIT solution was prepared as follows: Alkaline phosphatase was labeled in 100 nmol of DIT using the Alkaline Phosphatase Labelling Kit-NH2 (Dongjin Chemical Research Institute) to prepare the alkaline phosphatase-labeled DIT stock solution. Alkaline phosphatase-labeled DIT stock solution was prepared by diluting it 5000-fold with assay buffer (0.1 mol / L Tris-hydrochloric acid buffer, pH 7.5, containing 2 mmol / L magnesium chloride, 20 μmol / L zinc chloride, 0.1% bovine serum albumin, and 0.1% sodium azide). Anti-DIT mouse antibody solution was prepared by diluting anti-dibromotyrosine monoclonal antibody (Japan National Institute for Aging Control) to a concentration of 100 ng / mL with assay buffer. A 0.5 mmol / L benzoyl methyl phosphate solution was prepared by dissolving benzoyl methyl phosphate to a concentration of 0.5 mmol / L in enzyme reaction buffer (50 mmol / L 2-monoethanolamine-hydrochloric acid buffer, pH 9.5, containing 0.002% Triton X-100). A 0.001% gloss solution was prepared by dissolving gloss in gloss diluent (0.2 mol / L potassium dihydrogen phosphate aqueous solution) to a concentration of 0.001%.

[0128] 2. Construction of the standard curve for DIT

[0129] 100 μL of DIT standard solution was aliquoted into a 96-well plate immobilized with anti-mouse IgG rabbit antibody. Then, 25 μL of alkaline phosphatase-labeled DIT solution and 25 μL of anti-DIT mouse antibody solution were added and mixed. The mixture was incubated at 10°C for 2 hours. After washing four times with a washing buffer containing 0.02% Triton X-100 and 0.1% sodium azide, 100 μL of 0.5 mmol / L benzoyl methyl phosphate solution was aliquoted and incubated at room temperature for 2 hours. Then, 50 μL of 0.001% gloss solution was aliquoted, mixed, and placed in a bioluminescence assay apparatus (Ato Corporation). 150 μL of 1.0 mol / L sodium hydroxide solution was aliquoted into the bioluminescence assay apparatus, and the luminescence intensity was recorded from 8 to 9 seconds after aliquoting.

[0130] 3. Results of the DIT standard curve

[0131] The results of the DIT standard curve are shown in Table 6 and Figure 11 It can be seen that a good standard curve was generated, which enables the determination of DIT based on the CLEIA method.

[0132] [Table 6]

[0133] DIT (ng / mL) Luminous intensity B / Bo 0 8.81E+06 100.0％ 15 8.01E+06 90.9％ 50 7.41E+06 84.0％ 150 5.93E+06 67.3％ 500 2.52E+06 28.6％ 1500 6.62E+05 7.5％ 5000 1.52E+05 1.7％

[0134] B / B0 = Luminous intensity at each concentration / Luminous intensity at 0 concentration

[0135] Industrial availability

[0136] The identification method of the present invention can identify destructive thyroiditis by measuring at least one of monoiodotyrosine (MIT) and diiodotyrosine (DIT) in a sample. In particular, it can be used to differentiate therapeutically necessary destructive thyroiditis from Basel-Dürer's disease. Furthermore, monoiodotyrosine (MIT) and diiodotyrosine (DIT) can also be used as biomarkers for the diagnosis or identification of destructive thyroiditis.

[0137] This invention can be applied, for example, to fields such as medicine and pharmacy.

Claims

1. Use of a reagent for determining at least one of monoiodotyrosine and diiodotyrosine in the preparation of products for differentiating between destructive and non-destructive thyroiditis.

2. The use according to claim 1, wherein, The determination of monoiodotyrosine and diiodotyrosine was performed.

3. The use according to claim 1 or 2, wherein, The amount of at least one of monoiodotyrosine and diiodotyrosine is higher in patients with destructive thyroiditis than in non-patients or patients with non-destructive thyroiditis.

4. A kit for the differential diagnosis of destructive thyroiditis, characterized in that, The identification kit includes: a unit for determining at least one of monoiodotyrosine and diiodotyrosine in a sample collected from a subject; and a unit for comparing the obtained measurement value of the subject with the measurement value of at least one of monoiodotyrosine and diiodotyrosine in corresponding samples collected from non-affected individuals, individuals with non-destructive thyroiditis, and individuals with destructive thyroiditis.

5. The identification kit according to claim 4, wherein, The determination of monoiodotyrosine and diiodotyrosine was performed.

6. The identification kit according to claim 4 or 5, wherein, The amount of at least one of monoiodotyrosine and diiodotyrosine is higher in patients with destructive thyroiditis than in non-patients or patients with non-destructive thyroiditis.

7. A kit for the differential diagnosis of destructive thyroiditis, characterized in that, The identification kit includes: a unit for determining at least one of monoiodotyrosine and diiodotyrosine in a sample collected from a subject; and a unit for comparing the obtained measured value of the subject with a cutoff value for monoiodotyrosine and / or diiodotyrosine, the cutoff value being a value at which the subject is identified as having destructive thyroiditis rather than non-destructive thyroiditis when the measured value of monoiodotyrosine and / or diiodotyrosine in the sample exceeds the value.

8. The identification kit according to claim 7, wherein, The determination of monoiodotyrosine and diiodotyrosine was performed.

9. The identification kit according to claim 7 or 8, wherein, The amount of at least one of monoiodotyrosine and diiodotyrosine is higher in patients with destructive thyroiditis than in non-patients or patients with non-destructive thyroiditis.

Images