How to differentiate destructive thyroiditis from other conditions

Abstract

The object of the present invention is to provide a method for differentiating destructive thyroiditis. The present invention relates to a method for differentiating destructive thyroiditis comprising measuring at least one of monoiodotyrosine and diiodotyrosine in a sample.

Description

Technology Field

[0001] The present invention relates to a method for distinguishing 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 based on iodine contained in food and secretes them into the blood. There are two types of endocrine cells in the thyroid gland, and in the follicular lumen of the follicular cells, which make up the majority, tyrosine residues undergo iodination in the presence of hydrogen peroxide. In short, during the process of iodine organification, iodine binds to the carbons at the 3rd and 5th positions of tyrosine residues of thyroglobulin (Tg), producing monoiodotyrosine (MIT) and diiodotyrosine (DIT), which are precursors of thyroid hormones. Subsequently, MIT and DIT polymerize by peroxidase to produce triiodothyronine (T3), a thyroid hormone, and DIT polymerize to produce thyroxine (T4). Inside the follicular cells, iodinated Tg releases T3, T4, MIT, and DIT by proteolytic enzymes. Furthermore, T3 and T4 are secreted into the bloodstream, and iodine ions are released from MIT and DIT through the function of deiodinase enzymes and reused. Most of the thyroid hormones secreted into the bloodstream from the thyroid exist bound to proteins in the blood, but it is the free T3 (FT3) or free T4 (FT4) that are not bound to proteins that exert their effects. The primary functions of thyroid hormones are largely related to maintaining life, but they also extend to various fields such as the heart and skeletal muscles, metabolism, the nervous system, and development.

[0003] Destructive thyroiditis is a disease in which the destruction of thyroid follicles occurs due to causes such as stress from childbirth or surgery, certain medications, or food, leading to the leakage of thyroid hormones into the bloodstream and causing symptoms of thyrotoxicosis such as palpitations, weight loss, and excessive sweating. Destructive thyroiditis includes subacute thyroiditis, which is accompanied by pain, and painless thyroiditis, which is not. Unlike Graves' disease, the state of thyroid hormone excess in destructive thyroiditis is temporary; although hormone levels may drop and hypothyroidism may develop while the destroyed thyroid is recovering, recovery usually occurs within a few months.

[0004] In the case of subacute thyroiditis, when the affected area is unilateral and there is mild pain, differentiation from undifferentiated thyroid carcinoma or intracystic hemorrhage becomes important, and in the case of painless thyroiditis, differentiation from Graves' disease becomes important. In the case of destructive thyroiditis, spontaneous healing is common, but in cases of severe symptoms, corticosteroids may be administered in the case of subacute thyroiditis, and β-blockers in the case of painless thyroiditis. Differentiation from Graves' disease is particularly important, and if Graves' disease medication is administered to a patient with painless thyroiditis, not only is there a possibility of developing hypothyroidism, but it also becomes a major problem if side effects of the Graves' disease medication (especially agranulocytosis) occur.

[0005] As a method for differentiating between destructive thyroiditis and Graves' disease, it is common practice to additionally measure anti-TSH receptor antibodies (TRAb) when a patient's FT3 and FT4 are hypertrophic and thyroid-stimulating hormone (TSH) is 0.1 μIU / mL or less; if the result is positive, Graves' disease is diagnosed, and if negative, destructive thyroiditis is diagnosed. Although TRAb measurement is performed using a kit, the antibody measured as TRAb cannot be considered a monoclonal antibody, and since conditions such as idiopathic myxedema also show hypertrophic levels, problems remain regarding its properties and measurement method. Furthermore, TRAb can rarely be positive in painless thyroiditis and subacute thyroiditis (Non-patent Literatures 1–4).

[0006] Other methods for differentiating between destructive thyroiditis and Graves' disease are known as follows.

[0007] (i) FT3 / FT4 ratio

[0008] If the FT3 / FT4 ratio rises to the same degree, it is painless thyroiditis, and if the FT3 / FT4 ratio is 2.5 or higher, it is Graves' disease, but the sensitivity and specificity are low.

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

[0010] If blood flow is reduced in a thyroid echo test, it is painless thyroiditis; if it is increased, it is Graves' disease.

[0011] (iii) Inferior thyroid artery blood flow velocity

[0012] If the rate is low from normal, it is painless thyroiditis; if it is abnormally high from normal, it is Graves' disease. However, there are cases where low is seen in Graves' disease and high is seen in painless thyroiditis.

[0013] (iv) 123 I or 99m Tc Scintigraphy (Intake Rate)

[0014] 123 I or 99mIf the intake of Tc (which is taken through the same mechanism as iodine) is reduced, it is painless thyroiditis; if it increases from normal, it is Graves' disease. 99m Tc is a radioactive isotope of the operating order; the test cannot be canceled, and in many cases, differentiation is completed by the time it is produced. Meanwhile, 99m Tc has the advantage of not having a restriction on iodine intake, but it becomes impossible to distinguish when large amounts of iodine are consumed.

[0015] (v) Measurement of urinary iodine excretion

[0016] If total urinary iodine excretion increases, it is painless thyroiditis; if it does not increase, it is Graves' disease. However, the timing of measurement is limited as it is affected by diet.

[0017] As such, although various differential diagnosis methods have been developed for destructive thyroiditis, there is no method that can diagnose it simply and accurately.

[0018] However, as previously mentioned, MIT is a precursor of thyroid hormone, a substance in which iodine is coordinated to the meta position of the phenol ring of tyrosine. Two molecules combine to form 3,3'-diiodothyronine. Additionally, in the colloid of the thyroid, one additional molecule combines with diiodothyrosine to become triiodothyronine.

[0019] Also, as previously mentioned, DIT is a precursor of thyroid hormone, a substance in which iodine is coordinated to another meta position of the phenol ring of monoiodotyrosine. Diiodotyrosine regulates the function of iodide peroxidase. It also combines with monoiodotyrosine in the thyroid colloid to form triiodothyronine. When two molecules of diiodotyrosine combine, thyroxine is produced.

[0020] Regarding MIT and DIT, their use as anticancer agents was known (Patent Document 1), but it was not known that they are released into the bloodstream after the thyroid gland is destroyed. Prior art literature

[0021] Japanese Patent Publication No. Hei 7-116031

[0022] Hitoshi Ikeda "25. "Hyperthyroidism, Hypothyroidism," "Guidelines for Clinical Examinations by Diagnostic Group 2003 ~Toward Standardization of Medical Care~," Compiled by the Japanese Society of Laboratory Medicine, 107–110, 2003. Hiroshi Yoshimura: Approach to Thyroid Hormone Abnormalities, Journal of the Japanese Society of Internal Medicine, Vol. 103, No. 4 (April 10, 2014): 855–861. 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, 1997): 48–53. Ayuko Nakano et al.: Rapid Differential Diagnosis of Graves' Disease and Destructive Thyroiditis at Initial Examination, Dokkyo Journal of Medical Science, 36 (2), 105–108, 2009. The problem to be solved

[0023] The objective of the present invention is to provide a method for differentiating destructive thyroiditis. means of solving the problem

[0024] The inventors of the present invention, having conducted a study to solve the above problem, discovered that the amounts of monoiodotyrosine and diiodotyrosine in the blood, which were not known to be released outside the thyroid gland after the destruction of the thyroid gland, were surprisingly higher in patients with destructive thyroiditis compared to 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 whose TSH and FT4 levels are within the standard range or patients with Graves' disease, and thus completed the present invention.

[0025] That is, the present invention is as follows.

[0026] [1]

[0027] A method for differentiating destructive thyroiditis comprising measuring at least one of monoiodotyrosine and diiodotyrosine in a sample.

[0028] [2]

[0029] A method of differentiation described in item [1] above, in which both monoiodotyrosine and diiodotyrosine are measured.

[0030] [3]

[0031] A method of identification described in item [1] or [2] above, in which the sample is blood.

[0032] [4]

[0033] A method for differentiation described in any one of items [1] to [3], wherein at least one measurement of monoiodotyrosine and diiodotyrosine in a sample is performed using LC-MS with their stable isotopes as internal standard substances.

[0034] [5]

[0035] A biomarker for differentiating destructive thyroiditis, characterized by being either monoiodotyrosine or diiodotyrosine in the sample.

[0036] [6]

[0037] The biomarker described in item [5] above, characterized by being both monoiodotyrosine and diiodotyrosine.

[0038] [7]

[0039] A biomarker listed in item [5] or [6] above, in which the sample is blood.

[0040] [8]

[0041] A kit for differentiating destructive thyroiditis, characterized by comprising means for measuring at least one of monoiodotyrosine and diiodotyrosine in a sample taken from a subject, and means for comparing the obtained measurement of the subject with at least one measurement of monoiodotyrosine and diiodotyrosine in a corresponding sample taken from a non-affected person, an affected person with non-destructive thyroiditis, and an affected person with destructive thyroiditis.

[0042] [9]

[0043] A kit for differentiating destructive thyroiditis, characterized by including means for measuring at least one of monoiodotyrosine and diiodotyrosine in a sample taken from a subject, and means for comparing the obtained measurement value of the subject with a cutoff value of monoiodotyrosine and / or diiodotyrosine.

[0044]

[10]

[0045] A differentiation kit described in item [8] or [9] above, in which both monoiodotyrosine and diiodotyrosine are measured. Effects of the invention

[0046] According to the present invention, destructive thyroiditis can be differentiated. In particular, destructive thyroiditis can be differentiated from non-destructive thyroiditis diseases, such as Graves' disease, plummer's disease, and transient hyperthyroidism during pregnancy, particularly Graves' disease. Brief explanation of the drawing

[0047] Figure 1 is a diagram showing the concentration of monoiodotyrosine (MIT) in the serum of a patient with thyroid disease. Figure 2 is a diagram showing the concentration of diiodotyrosine (DIT) in the serum of a patient with thyroid disease. Figure 3 is a diagram showing the concentration of MIT in the serum of a patient with thyroid disease. Figure 4 is a diagram showing the results of Figure 3 in quartiles (box plot). Figure 5 is a diagram showing the concentration of DIT in the serum of a patient with thyroid disease. Figure 6 is a diagram showing the results of Figure 5 in quartiles (box plot). Figure 7 is a diagram combining the results of Figures 1 and 3 into one. Figure 8 is a diagram showing the results of Figure 7 in quartiles (box plot). Figure 9 is a diagram combining the results of Figures 2 and 5 into one. Figure 10 is a diagram showing the results of Figure 9 as quartiles (box plot). Figure 11 is a diagram showing the standard curve of DIT. Specific details for implementing the invention

[0048] In the present invention, "non-destructive thyroiditis disease" refers to thyroid diseases and thyroid dysfunction other than destructive thyroiditis as described above, and examples include Graves' disease, Plummer's disease, and transient hyperthyroidism during pregnancy. Furthermore, "non-destructive thyroiditis disease" also includes diseases in which the thyroid is totaled and levothyroxine (LT4) is administered, diseases in which abnormalities are observed in thyroid test results but the disease was not classified as a thyroid disease, and diseases in which thyroid hormone or TSH values ​​are within the normal range while the patient is being treated for thyrotoxicosis (hyperfunction) or hypofunction.

[0049] In the present invention, at least one amount of monoiodotyrosine and diiodotyrosine (hereinafter collectively referred to as the “biomarkers of the present invention”) in samples collected from destructive thyroiditis affected individuals, non-affected individuals, and non-destructive thyroiditis affected individuals is higher in destructive thyroiditis affected individuals compared to non-affected individuals and non-destructive thyroiditis affected individuals, so destructive thyroiditis can be distinguished by measuring the biomarkers of the present invention, and in particular, destructive thyroiditis can be distinguished from non-destructive thyroiditis (especially Graves’ disease).

[0050] Specifically, for example, at least one of the biomarkers of the present invention is measured in samples collected from a subject, a non-affected individual, and an individual with non-destructive thyroiditis disease, respectively, and by comparing the obtained measurement of the subject with the measurements of the non-affected individual and the individual with non-destructive thyroiditis disease, it is possible to differentiate whether the subject is affected by destructive thyroiditis. For example, if the measurement of the subject is significantly higher than the measurements of the non-affected individual and the individual with non-destructive thyroiditis disease by means of a statistical method, it can be differentiated that the subject is affected by destructive thyroiditis rather than non-destructive thyroiditis disease.

[0051] Alternatively, a cutoff value may be set and the obtained measurement of the subject may be compared with the cutoff value to differentiate destructive thyroiditis. The cutoff value refers to a value at which, if the measurement of MIT and / or DIT in the sample of the subject exceeds this value, the subject is identified as having destructive thyroiditis rather than non-destructive thyroiditis.

[0052] The cutoff value varies depending on the animal species or sample. For example, the cutoff value for human serum of MIT is preferably 250 pg / mL (e.g., 260 pg / mL), more preferably 210 pg / mL (e.g., 200 pg / mL), even more preferably 160 pg / mL, and particularly preferably 120 pg / mL. Also, for example, the cutoff value for human serum of DIT is 400 pg / mL, preferably 300 pg / mL (especially 350 pg / mL), more preferably 260 pg / mL, even more preferably 250 pg / mL, and particularly preferably 200 pg / mL.

[0053] The cutoff value can be calculated, for example, based on the ROC (Receiver Operating Characteristic) curve.

[0054] In the present invention, differentiation is made possible by measuring and comparing at least one of monoiodotyrosine and diiodotyrosine, but it is particularly preferable to measure and compare diiodotyrosine. Furthermore, in the present invention, since the precision and accuracy of differentiation are improved, it is preferable to measure and compare both monoiodotyrosine and diiodotyrosine.

[0055] In the method of differentiation of the present invention, “subject” refers to an animal including a human, “non-affected” refers to an animal including a human whose TSH and FT4 are within the standard range and who is not undergoing treatment for thyroid diseases such as thyrotoxicosis and hypothyroidism or thyroid dysfunction, “non-destructive thyroiditis affected” refers to an animal including a human diagnosed by a doctor or veterinarian as having non-destructive thyroiditis, and “destructive thyroiditis affected” refers to an animal including a human diagnosed by a doctor or veterinarian as having destructive thyroiditis.

[0056] In addition, the reference values ​​for the above TSH and FT4 should be values ​​that are not significantly abnormal compared to healthy humans or healthy animals other than humans, and although they vary depending on the animal species or sample, for example, the reference value for the above TSH in human serum can be 0.61 to 4.68 μIU / mL, and the reference value for the above FT4 in human serum can be 0.76 to 1.65 ng / dL.

[0057] Examples of animals other than humans include pets such as dogs, cats, and parakeets, livestock such as cows, pigs, and horses, and poultry such as chickens.

[0058] In the differentiation of the present invention, when comparing measurements of a subject, a non-affected individual, an affected individual with non-destructive thyroiditis, and an affected individual with destructive thyroiditis, or when comparing the measurements of a subject with a cutoff value, it is preferable that these values ​​be measured from at least the same animal species. Furthermore, it is preferable that conditions such as subspecies (including race), sex, age (including months and weeks), height, body weight, and samples are also identical.

[0059] In the present invention, the "sample" for which MIT or DIT is measured is collected from a subject, a non-affected person, a person with non-destructive thyroiditis, and a person with destructive thyroiditis. Any sample of biological origin other than the thyroid may be used without particular limitation. Examples include blood (whole blood, serum, plasma), saliva, tears, sweat, urine, feces, bile, tissue, hair, or living cells, or culture media of tissues or living cells, or preparations obtained from organs (provided that organs are excluded from the thyroid, and tissues and cells are excluded from the thyroid). Preferably, it is blood, saliva, and urine, and more preferably, blood, particularly serum and plasma.

[0060] In the present invention, the method for measuring the biomarker of the present invention is not particularly limited and conventional measurement methods may be used as long as it can measure monoiodotyrosine and / or diiodotyrosine in a sample.

[0061] For example, the radioimmunoassay (RIA) method (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 K. J Clin Endocrinol Metab. 1991 Apr ; 72 (4) : 945-53) or the LC-MS / MS method (Effects of exogenous monoiodotyrosine on the serum levels of anterior pituitary hormones. 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 immunoassay (ELISA), or electrochemiluminescent immunoassay (ECLIA) can also be used.

[0062] Preferably, liquid chromatography mass spectrometry (LC-MS) with a wide quantitative range that can be performed easily without the need for radioactive materials or derivatization treatment, and can also use stable isotopes as internal standard materials (IS).

[0063] LC-MS refers to an analysis method performed using a device that combines a liquid chromatograph and a mass spectrometer, and examples include liquid chromatography tandem mass spectrometry (LC-MS / MS) and liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS), and preferably LC-MS / MS.

[0064] To perform mass spectrometry, it is necessary to ionize the target component. Ionization methods include atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), and atmospheric pressure photoionization (APPI). By combining these ionization methods with LC-MS / MS, techniques such as Liquid Chromatography-Electrospray ionization based Tamdem Mass Spectrometry (LC-ESI / MS / MS) and Liquid Chromatography-Atmospheric pressure chemical ionization based Tamdem Mass Spectrometry (LC-APCI / MS / MS) are used. The measurements themselves can be performed using general methods. For this measurement, LC-ESI / MS / MS is preferred. Ion detection includes positive ion detection and negative ion detection; among these, negative ion detection is preferred for this measurement.

[0065] In addition, for the mass spectrometer, examples include magnetic field type, quadrupole type, and time-of-flight type, but in the present invention, it is preferable to use a quadrupole type, which has good quantitative accuracy, a wide dynamic range, and good linearity.

[0066] In addition, for the detection of ions in quantitative analysis, examples include Selected Ion Monitoring, which selectively detects only the target ion, and Selected Reaction Monitoring (SRM), which selects one of the ion species generated in the first mass spectrometer as a precursor ion and detects the product ion generated by the cleavage of that precursor ion in the second mass spectrometer. In the present invention, measurement by SRM is preferred, as the signal-to-noise ratio is improved by increasing selectivity and reducing noise.

[0067] Stable isotopes of monoiodotyrosine and diiodotyrosine are commercially available or can be synthesized by conventional methods.

[0068] The differentiation method of the present invention is superior in that, since MIT or DIT is the measurement target, the above-mentioned negative ion detection LC-ESI / MS / MS measurement method can be used, and destructive thyroiditis can be differentiated simply and accurately.

[0069] The present invention also relates to a method of using monoiodotyrosine or diiodotyrosine, preferably both, as a biomarker for differentiating destructive thyroiditis, in particular for differentiating destructive thyroiditis from non-destructive thyroiditis (particularly Graves' disease).

[0070] The present invention also relates to a identification kit for use in the identification method of the present invention.

[0071] The kit used for the differentiation method of the present invention may include at least one selected from a document describing a buffer solution, an acid, a base, an alcohol, a syringe, an antibody, and a measurement sequence. In addition to these, the kit used for the differentiation method of the present invention may further include a document describing a judgment criterion.

[0072] More specifically, the “differentiation kit” of the present invention includes means necessary for differentiating destructive thyroiditis by comparing at least one measurement of the biomarker of the present invention in a sample taken from a subject with at least one measurement of the biomarker of the present invention in a corresponding sample taken from a non-affected person, a person with non-destructive thyroiditis, and a person with destructive thyroiditis, or a cutoff value. For example, as at least one means of measuring the biomarker of the present invention present in a sample collected from a subject, a sample collection device or preparation device, a measuring device or reagent of MIT and / or DIT, such as a column cartridge, etc., may be provided. Additionally, as a means of comparing at least one measurement value of the biomarker of the present invention in a sample collected from a subject with at least one measurement value or cutoff value of the biomarker of the present invention in a corresponding sample collected from a non-affected person, a person with non-destructive thyroiditis disease, and a person with destructive thyroiditis disease, a comparison table of the respective measurement values ​​or cutoff values ​​of the non-affected person, a person with non-destructive thyroiditis disease, and a person with destructive thyroiditis disease may be provided. Among these, the comparison table is particularly useful as it makes it possible to differentiate the target disease without relying on a physician's judgment.

[0073] The present invention also relates to a screening method for a treatment or prophylactic agent for destructive thyroiditis, comprising measuring at least one, preferably both, of monoiodotyrosine and diiodotyrosine in a sample.

[0074] Examples

[0075] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples. In addition, the apparatus and materials used in the examples are as follows.

[0076] [Example 1] Differentiation of patients with destructive thyroiditis by LC-MS / MS measurement (1)

[0077] 1. Preparation of samples containing MIT and DIT

[0078] 0.05 mL of human serum and internal standard (300 pg / 0.05 mL of MIT-) in a test tube 13 C6 and DIT- 13 C9 15 0.05 mL of a mixed methanol solution of N was added, and 0.1 mL of a 25 mg / mL dithiothreitol / 1% pyridine aqueous solution was added. After stirring, the mixture was left to stand at room temperature for 30 minutes. After standing, 1 mL of purified water and 4 mL of methyl-tert-butyl ether were added to the test tube. After shaking for 5 minutes, the mixture was centrifuged for 3 minutes to remove the methyl-tert-butyl ether. 0.5 mL of acetic acid / acetonitrile (1:50) was added to the aqueous layer. After centrifuging for 3 minutes, the supernatant was transferred to another test tube and loaded into a HyperSep (registered trademark) CX cartridge (Thermo Fisher) that had been conditioned beforehand with 3 mL of methanol, 1 mL of purified water, and 1 mL of a 1% acetic acid aqueous solution. After washing with 1 mL of purified water and 3 mL of methanol, MIT and DIT were eluted with 1 mL of methanol / purified water / ammonia water (6:14:1), the eluent was removed by distillation using a centrifugal evaporator, and dissolved in 0.1 mL of a 20 mM aqueous ammonium formate solution to prepare a sample containing MIT and DIT.

[0079] 2. LC-MS / MS conditions

[0080] The sample prepared above was analyzed by LC-MS / MS. The conditions for LC-MS / MS are shown in Table 1 below.

[0081]

[0082] 3. Validation Results

[0083] The validation results of the above LC-MS / MS analysis are shown in Table 2 below.

[0084]

[0085] As shown in Tables 1 and 2, the measurement method of MIT and DIT by LC-MS / MS according to the present invention has a short measurement time of 3 minutes per sample, a lower quantification limit of 60 pg / mL, and good validation results.

[0086] Using the method of measuring MIT and DIT by LC-MS / MS of the present invention described above, MIT and DIT in human serum of patients with thyroid disease were measured. The results are shown in Table 3 and Figures 1 and 2.

[0087]

[0088] Control group: Patients who had physical abnormalities or data suggesting thyroid disease during a medical examination, and whose TSH and FT4 levels were within the standard range despite being measured.

[0089] DT: Patients diagnosed with destructive thyroiditis (DT) (1 subacute, 2 painless)

[0090] GD: Patients diagnosed with Graves' disease (GD)

[0091] GTH: Patients with transient hyperthyroidism during pregnancy

[0092] From Table 3 and Figures 1 and 2, it was found that destructive thyroiditis can be differentiated by measuring MIT or DIT in the blood. In particular, it was found that destructive thyroiditis can be differentiated from non-destructive thyroid diseases, especially Graves' disease.

[0093] [Example 2] Differentiation of patients with destructive thyroiditis by LC-MS / MS measurement (2)

[0094] MIT and DIT in human serum of patients with thyroid disease were measured according to the method described in Example 1. The results are shown in Table 4 and Figures 3 to 6.

[0095]

[0096] Control group: Patients with thyroids who had physical and data abnormalities suspected of thyroid disease during a physician's examination, and whose TSH and FT4 levels were within the standard range despite measurement.

[0097] DT: Patients diagnosed with destructive thyroiditis (DT)

[0098] GD: Patients diagnosed with Graves' disease (GD)

[0099] From Table 4 and Figures 3 to 6, it was found that destructive thyroiditis can be differentiated by measuring MIT or DIT in the blood. In particular, it was found that destructive thyroiditis can be differentiated from Graves' disease.

[0100] [Example 3] Differentiation of patients with destructive thyroiditis by LC-MS / MS measurement (3)

[0101] The results of Examples 1 and 2 were aggregated to calculate the data. The results are shown in Table 5 and Figures 7 to 10.

[0102]

[0103] Control group: Patients with thyroids who had physical and data abnormalities suspected of thyroid disease during a physician's examination, and whose TSH and FT4 levels were within the standard range despite measurement.

[0104] DT: Patients diagnosed with destructive thyroiditis (DT)

[0105] GD: Patients diagnosed with Graves' disease (GD)

[0106] GTH: Patients with transient hyperthyroidism during pregnancy

[0107] Table 5 and Figures 7 to 10 show the combined results of Examples 1 and 2, so it was found that the amount of data increased and the precision became higher.

[0108] From Table 5 and Figures 7 to 10, it was found that destructive thyroiditis can be differentiated by measuring MIT or DIT in the blood. In particular, it was found that destructive thyroiditis can be differentiated from non-destructive thyroid diseases, especially Graves' disease.

[0109] [Example 4] Measurement of DIT by Chemiluminescent Immunoassay (CLEIA)

[0110] 1. Preparation of plates and solutions

[0111] The preparation of the anti-mouse IgG rabbit antibody solidification 96-hole plate was carried out as follows. The anti-mouse IgG rabbit antibody solution (Jackson Immunoresearch, Inc.) was diluted to a concentration of 10 μg / mL with phosphate-buffered physiological saline, and 100 μL of the diluted solution was dispensed onto a 96-hole white plate (Thermo Fisher Scientific, Inc.). Next, after reacting at 4°C for 16 hours, the plate was prepared by washing twice with 0.02% Triton (Trademark) X-100 and additionally washing twice with 5% sucrose. The alkaline phosphatase-labeled DIT solution was prepared as follows. Using the Alkaline Phosphatase Labelling Kit-NH2 (Dojin Chemical Research Institute), 100 nmol of DIT was labeled with alkaline phosphatase to obtain the alkaline phosphatase-labeled DIT stock solution. The alkaline phosphatase-labeled DIT stock solution was prepared by diluting it 5000-fold with the measurement buffer (0.1 mol / L Tris-hydrochloride buffer pH 7.5 containing 2 mmol / L magnesium chloride, 20 μmol / L zinc chloride, 0.1% bovine serum albumin, and 0.1% sodium azidide). The anti-DIT mouse antibody solution was prepared by diluting the anti-dibromotyrosine monoclonal antibody (Japan Aging Control Research Institute) with the measurement buffer to a concentration of 100 ng / mL. The 0.5 mmol / L phenacyl phosphate solution was prepared by dissolving phenacyl phosphate at a concentration of 0.5 mmol / L in the enzyme reaction buffer (50 mmol / L 2-monoethanolamine-hydrochloride buffer pH 9.5 containing 0.002% Triton (registered trademark) X-100). A 0.001% lucigenin solution was prepared by dissolving lucigenin at a concentration of 0.001% in a lucigenin diluted solution (0.2 mol / L aqueous solution of potassium dihydrogen phosphate).

[0112] 2. Creation of the DIT Standard Curve

[0113] 100 μL of DIT standard solution was dispensed into a 96-hole plate containing solidified anti-mouse IgG rabbit antibody, and an additional 25 μL of alkaline phosphatase-labeled DIT solution and 25 μL of anti-DIT mouse antibody solution were added and mixed, and the mixture was reacted at 10°C for 2 hours. After washing 4 times with a washing solution containing 0.02% Triton (Trademark) X-100 and 0.1% sodium azidide, 100 μL of 0.5 mmol / L phenacyl phosphate solution was dispensed and the mixture was reacted at room temperature for 2 hours, after which 50 μL of 0.001% lucigenin solution was dispensed and mixed, and the mixture was set in a biochemiluminescence measuring device (Ato Co., Ltd.). 150 μL of a 1.0 mol / L sodium hydroxide solution was dispensed into a bio-chemiluminescence measuring device, and the luminescence intensity was accumulated from 8 seconds after dispensing to 9 seconds after dispensing.

[0114] 3. Results of the DIT Standard Curve

[0115] The results of the DIT standard curve are shown in Table 6 and Figure 11. A good standard curve was formed, and it was found that DIT could be measured by the CLEIA method.

[0116]

[0117] B / B0 = Luminous intensity at each concentration / Luminous intensity at concentration 0

[0118] Industrial applicability

[0119] According to the differentiation method of the present invention, destructive thyroiditis can be differentiated by measuring at least one of monoiodotyrosine (MIT) and diiodotyrosine (DIT) in a sample. In particular, it is possible to differentiate between destructive thyroiditis and Graves' disease, which is necessary for treatment. In addition, monoiodotyrosine (MIT) and diiodotyrosine (DIT) can be used as biomarkers for the diagnosis or differentiation of destructive thyroiditis.

[0120] The present invention can be used in fields such as medicine or pharmacy, for example.

Claims

Claim 1 A method for providing information for differentiating between a destructive thyroiditis affected body and a non-destructive or non-destructive thyroiditis affected body, comprising measuring at least one of monoiodotyrosine and diiodotyrosine in a sample. Claim 2 A method according to claim 1, wherein information for distinguishing a destructive thyroiditis affected body is provided based on whether at least one measurement of monoiodotyrosine and diiodotyrosine in a sample is (i) higher than that of a non-affected body or a non-destructive thyroiditis affected body, or (ii) higher than that of at least one cutoff value of monoiodotyrosine and diiodotyrosine. Claim 3 A method according to claim 1, wherein both monoiodotyrosine and diiodotyrosine are measured. Claim 4 A method according to claim 1, wherein the sample is blood or urine. Claim 5 A method according to any one of claims 1 to 4, wherein at least one measurement of monoiodotyrosine and diiodotyrosine in a sample is performed using an LC-MS that uses their stable isotopes as internal standard substances. Claim 6 A composition for differentiating between a destructive thyroiditis affected person and a non-affected person or a non-destructive thyroiditis affected person, comprising either monoiodotyrosine or diiodotyrosine in a sample as a biomarker. Claim 7 A composition for differentiating a destructive thyroiditis patient based on whether the measured value of a biomarker is (i) higher than that of a non-disruptive or non-destructive thyroiditis patient, or (ii) higher than the cutoff value of monoiodotyrosine or diiodotyrosine. Claim 8 A composition according to claim 6, comprising both monoiodotyrosine and diiodotyrosine as biomarkers. Claim 9 A composition according to any one of claims 6 to 8, wherein the sample is blood or urine. Claim 10 A kit for differentiating a destructive thyroiditis affected body and a non-affected body or a non-destructive thyroiditis affected body, characterized by comprising means for measuring at least one of monoiodotyrosine and diiodotyrosine in a sample taken from a subject, and means for comparing the obtained measurement of the subject with at least one measurement of monoiodotyrosine and diiodotyrosine in a corresponding sample taken from a non-affected body, a non-destructive thyroiditis affected body, and a destructive thyroiditis affected body. Claim 11 A kit according to claim 10 for differentiating a destructive thyroiditis patient based on whether the obtained measurement of the subject is higher than at least one measurement of monoiodotyrosine and diiodotyrosine in a corresponding sample taken from a non-disruptive thyroiditis patient or a non-disruptive thyroiditis patient. Claim 12 A kit for differentiating between a destructive thyroiditis patient and a non-destructive thyroiditis patient, characterized by including means for measuring at least one of monoiodotyrosine and diiodotyrosine in a sample taken from a patient, and means for comparing the obtained measurement of the patient with a cutoff value of monoiodotyrosine and / or diiodotyrosine. Claim 13 A kit according to claim 12 for differentiating destructive thyroiditis patients based on whether the obtained measurement of the subject is higher than the cutoff value of monoiodotyrosine and / or diiodotyrosine. Claim 14 A kit in which both monoiodotyrosine and diiodotyrosine are measured in any one of claims 10 to 13.

Images