JP6937607B2 - Method for detecting thyroid disease by PSPLA1, method for providing information and reagents - Google Patents

Description

The present invention relates to a method for detecting thyroid disease by measuring the concentration of phosphatidylserine-specific phospholipase A1 in a human sample, a method for providing information for determining a therapeutic effect, and a reagent thereof.

For the diagnosis and management of hyperthyroidism, thyroid hormone and thyroid stimulating hormone, which receives negative feedback from thyroid hormone, are used. The optimal thyroid hormone level varies depending on the individual patient, and even if it slightly exceeds the standard range, there is no problem, and conversely, even if it is within the standard range, thyroid hormone is optimal for the relevant patient. In some cases, it may be difficult to determine the symptoms based on the general level of thyroid hormone. Thyroid-stimulating hormone, which receives negative feedback from thyroid hormone, is often measured at the same time, but fluctuations in thyroid-stimulating hormone reflect thyroid function in real time because there is a time lag of 2 weeks to several months from the action of thyroid hormone. There is a problem of not doing it. In addition, anti-thyroid stimulating hormone antibody (hereinafter, sometimes referred to as TRAb), which is one of the causative agents of Graves'disease, is also effective for diagnostic discrimination of Graves' disease, but the fluctuation after intervention is thyroid hormone. Since there is a further time lag than thyroid stimulating hormone, it is not suitable for grasping the state of thyroid function in real time. Therefore, alkaline phosphatase and total cholesterol, which reflect the status of thyroid hormone, may be used as auxiliary test items for thyroid function. These test items are used as indicators for suspecting thyroid disease, but the thyroid gland. There is a limit to the index of thyroid function because it fluctuates greatly not only by disease but also by other factors such as lifestyle.

In clinical practice, thyroid hormone is an item to be examined based on common medical examination results abnormalities such as dyslipidemia, hypolipidemia, agitation, and tremor, and complaints from patients. In many cases, thyroid disease is confirmed by additional tests such as additional blood tests, diagnostic imaging by radioiodine uptake, ultrasonography, and biopsy. Therefore, there is an urgent need for a simple test method that enables differential diagnosis between hyperthyroidism represented by Graves' disease and healthy subjects who deviate from the reference range.

Furthermore, in Graves' disease, therapeutic intervention with antithyroid drugs is typical, but the medication is continued with the normalization of thyroid hormone as a guide, and the normalization of thyroid stimulating hormone after the normalization of thyroid hormone is awaited. At this time, treatment is advanced while reducing the dosage, but since there is no index to control the dosage for several weeks to several months from normalization of thyroid hormone to normalization of thyroid stimulating hormone, thyroid function due to overdose. There is also a demand for markers that reflect thyroid function in real time, as there are cases of excessive hypothyroidism.

Phosphatidylserine-specific phospholipase A1 is a protein belonging to the lipase family identified in 1997, and hydrolyzes the acyl group at the sn-1 position using phosphatidylserine, which is a serine lipid having a serine residue on the polar head, as a specific substrate. (Non-Patent Document 1). This enzyme is mainly produced by activated platelets and macrophages, and its involvement in the inflammatory response by mast cell activation has been known for a long time. However, since the lysophosphatidylserine receptor was recently identified, lysophosphatidylserine It is assumed that it is involved in various cell function regulation through the receptor. It is also suggested that it is involved in the control of the coagulation system by removing phosphatidylserine exposed on the surface of platelet cells and the control of dead cell processing such as the removal of phosphatidylserine on the surface of apoptotic cells, but it is still unclear. There are also many.

There are few reports of diseases in which the serum phosphatidylserine-specific phospholipase A1 concentration fluctuates, and the concentration is increased in systemic lupus erythematosus (Patent Document 1), increased in epilepsy, decreased in leukemia, and septicemia model as an animal model. Although there are reports of increased serum concentration in mice and increased expression in ischemic brain disease model rat brain tissue (Patent Document 2), there are no reports showing a relationship with thyroid disease.

The measurement of phosphatidylserine-specific phospholipase A1 itself is possible by, for example, the method described in Non-Patent Document 2 or Patent Document 2.

Japanese Patent No. 5625155 Japanese Patent No. 5288712

J. Biol. Chem. 1997; 2192-2198 Clin. Chim. Acta 2010; 411: 1090-1094

So far, it has been reported that the blood concentration of phosphatidylserine-specific phospholipase A1 fluctuates in some diseases, but there is no report on its association with thyroid disease. The present invention is a method for detecting thyroid disease by measuring the phosphatidylserine-specific phospholipase A1 concentration, a method for providing information for determining the therapeutic effect at the time of intervention for thyroid disease, and a dosage based on the determination. Is to provide a way to control.

The present inventors measured the phosphatidylserine-specific phospholipase A1 concentration in the blood of patients with various endocrine diseases, and as a result of repeated studies, the phosphatidylserine-specific phospholipase A1 concentration in the blood increased in thyroid disease. We have found that the concentration decreases with the improvement of the medical condition by the intervention, and arrived at the present invention.

That is, the present invention includes the following inventions:
(1) A method for detecting thyroid disease, which comprises measuring phosphatidylserine-specific phospholipase A1 in a human sample.
(2) A method for providing information for determining the therapeutic effect of a patient with thyroid disease.
Measuring phosphatidylserine-specific phospholipase A1 in the patient-derived specimen, and
To associate the measured value with the therapeutic effect of the patient,
How to include.
(3) The method according to (1) or (2), wherein the thyroid disease is hyperthyroidism or thyroiditis.
(4) The method according to (3), wherein hyperthyroidism is Graves' disease.
(5) The method according to any one of (1) to (4), wherein the phosphatidylserine-specific phospholipase A1 is measured by an immunochemical method using an antibody that recognizes the phosphatidylserine-specific phospholipase A1.
(6) Information for detecting the disease or determining the therapeutic effect by the method according to any one of (1) to (5), which contains an antibody that recognizes phosphatidylserine-specific phospholipase A1. Reagents for providing.

According to the present invention, it is possible to detect thyroid disease by measuring phosphatidylserine-specific phospholipase A1 in a human sample, and it is also possible to determine the effect after intervention. Human phosphatidylserine-specific phospholipase A1 can be quantified in a short time by performing measurement using the immunological quantification reagent described in Patent Document 2 and the like, and can be easily and inexpensively tested even in a small-scale medical facility. Reagents can be provided.

It is a figure which shows the distribution of the phosphatidylserine-specific phospholipase A1 measurement value of the healthy person and the untreated Graves' disease patient sample. The display of the boxplot is a typical notation, specifically, the central box shows the median value of the 25-75th percentile, and the upper and lower bar lines are 1.5 times or more of the 75th and 25th percentile values. Shows the maximum and minimum values excluding the data in. It is a figure which shows the distribution of the phosphatidylserine-specific phospholipase A1 measurement value of the untreated Graves'disease and the therapeutic intervention Graves' disease patient sample. Treatment intervention The group in which either FT4 or FT3 is positive is indicated by a plus symbol, and the group in which TRAb is positive is indicated by a plus symbol. It is a figure which shows the distribution of the measured value. It is a figure which shows the distribution of the phosphatidylserine-specific phospholipase A1 measurement value of the healthy person and the untreated thyroiditis patient sample.

The sample used in the present invention is not particularly limited, and blood components and the like can be mentioned. Among them, whole blood, blood cells, plasma and the like are preferable, and serum and plasma are more preferable.

In the present invention, the thyroid disease is not particularly limited, and examples thereof include Graves' disease, which is hyperthyroidism, and thyroiditis.

In the present invention, the method for measuring phosphatidylserine-specific phospholipase A1 is not particularly limited, and examples thereof include an immunochemical method using an antibody that recognizes phosphatidylserine-specific phospholipase A1. More specifically, it can be carried out using an immunological measurement reagent containing an antibody that recognizes phosphatidylserine-specific phospholipase A1. At this time, for example, a reagent containing a immobilized antibody that recognizes the phosphatidylserine-specific phospholipase A1 to be measured and a labeled antibody such as an enzyme that recognizes the phosphatidylserine-specific phospholipase A1 to be measured at a site different from the immobilized antibody is used. It can be carried out by the sandwich method. For example, the reagents described in Non-Patent Document 2 and Patent Document 2 can be mentioned.

The present invention makes it possible to detect thyroid disease by measuring phosphatidylserine-specific phospholipase A1 in a human sample. Specifically, when the measured value of phosphatidylserine-specific phospholipase A1 in a human sample is higher than the measured value of a healthy person, the patient has thyroid disease, that is, the thyroid disease can be detected.

Further, the present invention is a method for providing information for determining a therapeutic effect of a patient with thyroid disease, in which phosphatidylserine-specific phospholipase A1 in a sample derived from the patient is measured, and the measured value and the patient. It is a method including associating with the therapeutic effect of. At this time, if the measurement is performed multiple times before and after the treatment intervention for thyroid disease or after the treatment intervention and the concentration of phosphatidylserine-specific phospholipase A1 is lower than that at the time of the previous measurement, it can be considered to have a therapeutic effect. .. The doctor determines the therapeutic effect with reference to the information provided by the present invention. That is, the method itself of the present invention does not include the final judgment act of determining the therapeutic effect, but includes the stage of providing the judgment material to the doctor.

Examples are shown below, but the present invention is not limited to the examples described in the examples. The following experiments were conducted with the approval of the Research Ethics Committee of each institution. Phosphatidylserine-specific phospholipase A1 (hereinafter referred to as PSPLA1) concentration measurement was carried out by preparing an immunological measurement reagent by the sandwich method in the same manner as in Patent Document 2 and using an automatic immunoassay device AIA series (manufactured by Tosoh Corporation). bottom.

Example 1: The number of patients, the number of samples, and the age of the subjects whose PSPLA1 concentration is measured are shown in Table 1. The difference between the number of patients with Basedou disease (50 untreated sera and 63 sera after intervention) and the number of thyroiditis is the same as the number of patients who are performing measurements before and after the intervention. On the other hand, the number of samples is exceeded. Table 2 shows a list of measured concentrations of PSPLA1 in the serum of the subjects. The upper limit of the 95th percentile of the PSPLA1 concentration in 52 healthy subjects by this measurement was 27.7 μg / L.

実施例２：健常者ならびにバセドウ病のＰＳＰＬＡ１濃度比較
実施例１で測定した健常者血清５２検体と未治療バセドウ病患者血清５０検体に分け、ＰＳＰＬＡ１測定値を比較した結果を図１に示す。また、未治療バセドウ患者血清５０検体と治療介入されたバセドウ患者血清６３検体のＰＳＰＬＡ１測定値を比較した結果を図２に示す。

Example 2: Comparison of PSPLA1 concentrations of healthy subjects and Graves'disease FIG. 1 shows the results of comparing the PSPLA1 measured values of 52 healthy subjects and 50 untreated Graves' disease patient sera measured in Example 1. In addition, FIG. 2 shows the results of comparing the PSPLA1 measured values of 50 untreated Graves'sera sera and 63 sera of Graves' disease who had undergone treatment intervention.

Significant differences were confirmed between the healthy subject group and the untreated Graves'disease group, and between the untreated Graves'disease group and the treatment intervention Graves' disease group, as shown in Table 3. Furthermore, the AUC (area under the curve), sensitivity, and specificity are calculated by ROC (Receiver Operating Characteristic) analysis of the discrimination ability between the healthy subject group and the untreated Basedow disease group, and between the untreated Basedow disease group and the therapeutic intervention Basedow disease group. The results are shown in Table 4. The AUC, sensitivity, and specificity between the healthy subject group and the untreated Graves' disease group showed high values, indicating that they have excellent discrimination ability. It was also shown to have sufficient discriminative ability in terms of AUC, sensitivity, and specificity between the untreated Graves'disease group and the interventional Graves' disease group.

実施例３：治療介入バセドウ病における治療効果に伴うＰＳＰＬＡ１濃度変動
実施例１で測定した治療介入したバセドウ病患者検体において、同時に測定したＦＴ４及びＦＴ３ならびにＴＲＡｂより４群に分類し、ＰＳＰＬＡ１濃度を検証した。ＦＴ４は１．６３ｎｇ／ｄＬ以下を陰性、ＦＴ３は３．８０ｐｇ／ｍＬ以下を陰性、ＴＲＡｂは２．０ＩＵ／Ｌ以下を陰性とし、その値を超えるものを陽性とし、ＦＴ４あるいはＦＴ３いずれかが陽性ならばＦＴ４／ＦＴ３は陽性群とした。
その濃度分布を図３に示す。ＦＴ４／ＦＴ３ならびにＴＲＡｂの陰性化に伴いＰＳＰＬＡ１濃度が低値を示す結果であった。

Example 3: Fluctuation of PSPLA1 concentration associated with therapeutic effect in therapeutic intervention Graves'disease The therapeutically-intervened Graves' disease patient sample measured in Example 1 was classified into 4 groups from FT4, FT3 and TRAb measured at the same time, and the PSPLA1 concentration was verified. bottom. FT4 is negative for 1.63 ng / dL or less, FT3 is negative for 3.80 pg / mL or less, TRAb is negative for 2.0 IU / L or less, positive for those exceeding that value, and positive for either FT4 or FT3. If so, FT4 / FT3 was regarded as a positive group.
The concentration distribution is shown in FIG. The result was that the PSPLA1 concentration showed a low value as FT4 / FT3 and TRAb became negative.

Example 4: Comparison of PSPLA1 concentrations of healthy subjects and thyroiditis FIG. 4 shows the results of comparing the PSPLA1 measured values of 52 healthy subjects and 7 untreated thyroiditis sera measured in Example 1. A significant difference was confirmed between the healthy subject group and the untreated thyroiditis group as shown in Table 5. Furthermore, Table 6 shows the results of calculating the AUC, sensitivity, and specificity of the discrimination ability between the healthy subject group and the untreated thyroiditis group by ROC analysis. The AUC, sensitivity, and specificity between the healthy subject group and the untreated thyroiditis group showed high values, indicating that they have excellent discrimination ability.

実施例５：ＰＳＰＬＡ１濃度と各種パラメーターとの相関性の検証
実施例１のバセドウ病患者におけるＰＳＰＬＡ１測定値と、甲状腺疾患に関連する各種パラメーターとの、相関性を検証した結果を表７に示す。一部、欠損値があるため使用した測定例数を表中に示している。その結果、ＦＴ３ならびにＦＴ４に対して比較的良い相関性が認められるものの、他のパラメーターとの相関性は非常に弱いものであった。

Example 5: Verification of Correlation between PSPLA1 Concentration and Various Parameters Table 7 shows the results of verification of the correlation between the PSPLA1 measured value in the Graves' disease patient of Example 1 and various parameters related to thyroid disease. The number of measurement examples used is shown in the table because there are some missing values. As a result, although a relatively good correlation was observed with FT3 and FT4, the correlation with other parameters was very weak.

Claims (4)

A method of measuring phosphatidylserine-specific phospholipase A1 in human serum and presenting the measured value as an index of Graves' disease or thyroiditis. A method for providing information for determining the therapeutic effect of a Graves'disease or thyroiditis patient, in which phosphatidylserine-specific phosphorlipase A1 in the serum derived from the patient is measured , and the measured value is used for the Graves' disease or thyroiditis patient. A method of providing information for determining the therapeutic effect of Graves' disease. The method according to claim 1 or 2, wherein the phosphatidylserine-specific phospholipase A1 is measured by an immunochemical method using an antibody that recognizes the phosphatidylserine-specific phospholipase A1. A reagent for use in the method according to claim 3, which contains an antibody that recognizes a phosphatidylserine-specific phospholipase A1.

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