KR102025992B1 - Lipid marker for identifying Graves’ disease and Graves’ disease with ophthalmopathy - Google Patents

Abstract

The present invention relates to a lipid marker for identifying Graves 'disease (GD) and Graves' eye disease (GO) and a method for providing information for identifying Graves 'disease (GD) and Graves' eye disease (GO) using the lipid marker.
In the present invention, selected lipid markers are selectively detected in a biological sample in a short time, so that Graves' disease (GD) disease and Graves' eye disease (GO) disease can be detected more quickly before external changes to the eyes of Graves' eye disease (GO) patients appear. Can be identified.

Description

Lipid marker for identifying Graves ’disease and Graves’ disease with ophthalmopathy}

The present invention relates to a lipid marker for identifying Graves 'disease (GD) and Graves' eye disease (GO) and a method for providing information for identifying Graves 'disease (GD) and Graves' eye disease (GO) using the lipid marker.

Graves' disease (GD), a hyperthyroidism, is an autoimmune disease in which the thyroid stimulating immunoglobulin activates the thyroid receptors (receptors), causing excess thyroid hormones. About 50% of people with GD are classified as Graves' disease with ophthalmopathy (GO), which causes increased orbital fat and connective tissue.

Diagnosis of Graves' disease (GD) is lower than normal by measuring the concentration of thyroid-stimulating hormones in the blood, which are involved in the role of thyroid hormone secretion. ) And T4 (tryroxine) is higher than normal.

In Graves 'eye disease (GO), which causes symptoms of eye changes, the hormone concentration is not different from that of GD, so diagnosis is made in the same way as Graves' disease (GD).

Currently, the identification of Graves' disease (GD) and Graves' eye disease (GO) is divided according to the measurement of the presence or absence of eye changes, there is a problem that early diagnosis of Graves' eye disease (GO) is difficult.

Thus, there is a need for new methods for identifying Graves 'disease (GD) and Graves' eye disease (GO).

1. Original Articles: Diagnostic Significance of TSH Measurement in Thyroid and Non-Thyroid Diseases, Korean Journal of Medicine, 40, 3, pp. 358-364, 1991

The present invention aims to establish lipid markers for the identification of Graves 'disease (GD) and Graves' eye disease (GO).

The present invention provides markers for Graves 'disease (GD) and Graves' eye disease (GO) comprising at least one selected from the group consisting of lysophosphatidylcholine (LPC) and triacylglycerol (TG).

In addition, the present invention comprises the steps of measuring the concentration of the lipid component in a biological sample isolated from the subject; And

And comparing the measured concentrations of lipid components and concentrations of lipid components isolated from the normal group to determine whether Graves 'disease (GD) and Graves' eye disease (GO).

The lipid component provides a method of identifying Graves 'disease (GD) and Graves' eye disease (GO) comprising at least one selected from the group consisting of lysophosphatidylcholine (LPC) and triacylglycerol (TG).

In the present invention, using ultra-high performance liquid chromatography-electrospray ionization-mass spectrometry (nLC-ESI-MS / MS), the detection limit is present in trace amounts at the level of femtomol (fmol / μl) or the detection intensity of the mass spectrometer is Low detection of various lipid species and structural analysis are possible. In particular, the analytical method has the advantage of being able to analyze lipids in a small amount of only 100 μl and 1 mL, respectively, in plasma and urine samples.

In addition, the present invention selectively detects selected lipid markers in a biological sample in a short time to detect Graves' disease (GD) disease and Graves' eye disease (GO) disease before external changes appear in the eyes of Graves' eye disease (GO) patients. It can be identified faster.

Accordingly, the technique of the present invention is expected to be developed into a future diagnostic method in which only a marker is selectively detected and diagnosed.

Figure 1 is a schematic diagram showing the process of securing lipids using the Folch modified with MTBE / CH ₃ OH extraction method.
2 is a schematic diagram showing an ultra-high performance liquid chromatography-electrospray ionization-mass spectrometer (nLC-ESI-MS / MS) system.
3 is a graph showing the results of comparing the ionization efficiency of various ionization mediators.
Figure 4 is a CID spectra graph of S1P analyzed S1P in a) cation mode and b) anion mode.
FIG. 5 is a graph showing PCA of statistically significant lipids in a) plasma and b) urine samples.
6 is a graph showing the change (normal group control) of lipid markers for identifying GD and GO selected from a) plasma samples and b) urine samples.

Hereinafter, the present invention will be described in more detail.

Lipids mentioned in the present invention are shown in Table 1 below.

Geological name Geological name Lysophosphatidylcholine
(lysophosphatidylcholine) LPC Sphingosine-1-phosphate
(sphingosine-1-phosphate) S1P Phosphatidylcholine
(phosphatidylcholine) PC Reeffingo Myelin
(Lysosphingomyelin) LSM Lysophosphatidylethanolamine
(lysophosphatidylethanolamine) LPE Sphingomyelin
(Sphingomyelin) SM (Phosphatidylethanolamine)
phosphatidylethanolamine PE Ceramide
(Ceramide) Cer PE plasmaogen
(PE plasmalogan) Pep Monohexyl ceramide
(Monohexosylceramide) MHC Lysophosphatidylglycerol
(lysophosphatidylglycerol) LPG Dihexyl ceramide
(Dihexosylceramide) DHC Phosphatidylglycerol
(phosphatidylglycerol) PG Trihexyl ceramide
(Trihexosylceramide) THC Lysophosphatidyl inositol
(lysophosphatidylinositol) LPI Diacylglycerol
(diacylglycerol) DG Phosphatidyl Inositol
(phosphatidylinositol) PI Triacylglycerol
(triacylglycerol) TG Phosphatidine acid
(phosphatidic acid) PA

The numbers described together with the lipid names used in the present invention represent the number of carbons and the number of multiple bonds in the lipids. For example, "16: 1-LPC" refers to lysophosphatidylcholine having 16 carbons and one multiple bond. Hereinafter, unless otherwise defined, the same can be applied to other lipids in the present invention.

In the present invention, "identification of Graves' disease (GD) and Graves' eye disease (GO)" not only distinguishes Graves' disease (GD) patients and Graves' eye disease (GO) patients, but also normal people and Graves' disease (GD) patients and Graves. It may include distinguishing a patient with ocular disease (GO). It may also include distinguishing between normal people and Graves' eye disease (GO) patients.

In this case, a normal person or a normal group refers to a person who has no symptoms and signs clinically suggestive of hyperthyroidism, and no symptoms and signs of thyroid ophthalmopathy, and does not have thyroid abnormalities in a blood test.

The present invention relates to markers for Graves 'disease (GD) and Graves' eye disease (GO) comprising at least one selected from the group consisting of lysophosphatidylcholine (LPC) and triacylglycerol (TG).

In the present invention, lysophosphatidylcholine (LPC) may be at least one selected from the group consisting of 16: 1-LPC and 18: 1-LPC, triacylglycerol (TG) is 44: 2-TG, 54: 7-TG, 60 At least one selected from the group consisting of: 13-TG, 46: 2-TG, 48: 1-TG, 50: 1-TG, 50: 2-TG, and 52: 0-TG.

The inventors have taken samples from patients' biological samples, ie blood (plasma) or urine, to extract lipids and find lipids for markers for identification of Graves' disease (GD) and Graves' eye disease (GO), and ultra high performance liquid chromatography Electrospray ionization-mass spectrometry (nLC-ESI-MS / MS) was used to compare and analyze lipid profile differences between Graves 'disease (GD) patients and Graves' eye disease (GO) patients. (GD) and Graves ophthalmopathy (GO) were identified and marker discovery studies were performed to diagnose older Graves ocular disease (GO) patients.

A total of 281 lipids were identified for this purpose. Molecular structures were identified from the CID mass spectrometry obtained during the nLC-ESI-MS / MS analysis, and individual quantification of each molecule in the plasma samples of normal, GD and GO patients was performed. 44: 2-TG Was increased by 2.06 times in the GD disease group and 2.99 times in the plasma of GO disease group, compared with the normal group, and the difference between the two groups was found to be 1.45 times statistically significant. 54: 7-TG increased 1.85-fold in plasma in GD group and 2.63-fold in GO group compared to normal group, showing a clear difference (1.42-fold) between the two disease groups, and 60: 13-TG. The difference between groups was 1.43 times of increase and decrease.

In addition, a total of 191 lipids in urine samples of normal group, GD disease and GO disease were individually quantified by SRM method using nLC-ESI-MS / MS. As a result, the 16: 1 LPC increased 3.65 times in the GD group and 6.12 times in the GO group compared to the normal group, showing a clear difference (1.68 times) in the GD group and the GO group. In the GD and GO groups, 7.30-fold and 9.99-fold increase, respectively, showed a significant difference (1.37-fold) between the two groups. In addition, 46: 2-TG was 0.60 times, 48: 1-TG was 0.66 times, 50: 1-TG was 0.63 times, 50: 2-TG was 0.68 times, and 52: 0-TG was 0.70 times. Statistically significant differences were found.

On the other hand, d18: 1-S1P increased 1.30 times in the GD group and 1.98 times in the GO group compared to the normal group in the plasma sample, and there was a difference of 1.53 times in the two groups, and 1.24 times in the GD group and the GO group in the urine sample. In 1.88 times, the difference between the two groups was 1.52 times.

Based on the results, lipids capable of identifying GD and GO in plasma and urine samples were selected as markers.

The markers are markers of Graves 'disease (GD), a hyperthyroidism, and are particularly specific for Graves' eye disease (GO), in which the orbital fat and connective tissue increase in about 50% of people with GD, resulting in increased eyeball. That is, the marker enables the identification of Graves 'disease (GD) and Graves' eye disease (GO).

Therefore, in the present invention, using the above-described marker, Graves' disease (GD) and Graves' eye disease (GO) disease before the external changes appear in the eyes of Graves' eye disease (GO) patients by selective detection in a biological sample quickly. Can be identified faster and early diagnosis of GO is possible.

As used herein, the term “diagnosis” refers to determining the susceptibility of an object to a particular disease or condition, determining whether an object currently has a particular disease or condition, or having a particular disease or condition. Determining the prognosis of an object, or therametrics (eg, monitoring the condition of an object to provide information about treatment efficacy).

The present invention also relates to a method for identifying Graves 'disease (GD) and Graves' eye disease (GO).

The identification method may include measuring a concentration of a lipid component in a biological sample isolated from a subject; And

The method may include determining whether Graves 'disease (GD) and Graves' eye disease (GO) by comparing the concentration of the lipid component and the concentration of the lipid component separated from the normal group.

In this case, the lipid component may be the aforementioned marker. In addition, the subject or subject may be a patient or prospective patient with symptoms associated with GO or GD, specifically those with clinically suggestive hyperthyroidism (thyroidoma, nerve hypersensitivity, fever hypersensitivity, weight loss, deep vein, etc.), And the symptoms and signs of thyroid ophthalmopathy (eye protrusion, diplopia, periorbital edema, back eyelid pull, etc.). In addition, the normal group refers to a group having no symptoms and signs indicating the above-mentioned clinically hypothyroidism, and no symptoms and signs of thyroid ophthalmopathy, and a blood test without thyroid abnormalities.

In one embodiment, a biological sample may be tissue, blood, or urine, and blood or urine may be used in the present invention. The blood may use plasma.

The lipid component in the present invention can be obtained from a subject through a method conventionally used in the art.

Securing complete lipids through the lipid extraction method is necessary to ensure the reliability of the data. If the effective lipid extraction method is not applied to various types of lipids, not only the target lipids may be extracted but also a matrix of various proteins, minerals, and ions in addition to the lipids may interfere with the ionization of the lipids. Thus, the sensitivity and selectivity of the lipid analysis are degraded. Therefore, among the various lipid extraction methods, a suitable method should be selected according to the type of lipid to be analyzed or the purpose of the experiment.

The most widely used method of extracting lipids is the Folch method, which uses chloroform, which is highly toxic and occupies a lower layer when mixed with an aqueous solution. The disadvantage is that it is difficult to recover.

Therefore, in the present invention, lipids can be extracted by modifying the Foltz method using methyl tert-butyl ether (MTBE), which is less toxic than chloroform instead of chloroform. In the present invention, the method may be referred to as a Foltz modified extraction method (Folch modified with MTBE / CH ₃ OH extraction method). The strain modified extraction method uses a solvent mixed with MTBE and methanol, and has an advantage that the organic solvent layer is on the upper layer due to the relatively low density of MTBE when the aqueous layer and the organic solvent layer are separated. In addition, the Poletz strain extraction method can extract lipid components with high recovery from relatively strong lysolipids and S1P to non-polar TG.

In the present invention, the concentration of the extracted lipid component can be measured using a chromatography / mass spectrometer.

Chromatography in the present invention may be used for quantitative liquid chromatography commonly used in the art, specifically capillary liquid chromatography (capillary LC), ultra high performance liquid chromatography (UPLC) or ultra high performance liquid chromatography (nUPLC) Or nLC). In addition, the mass spectrometer may use a mass spectrometer capable of performing tandem mass spectrometry in an electrospray ionization (ESI) mass spectrometer commonly used in the art. Specifically, an ion trap MS, an ion trap MS, a triple quadrupole MS, a Q-TOF or an orbit lab MS may be used as the mass spectrometer.

In the present invention, lipids can be measured using an ultra-high performance liquid chromatography-electrospray ionization-mass spectrometer (nLC-ESI-MS / MS).

The ultra-high performance liquid chromatography-electrospray ionization-mass spectrometer can be used to analyze and compare lipids in biological samples within about 30 minutes to identify two diseases, GD and GO.

In the present invention, it is possible to identify Graves' disease (GD) and Graves' eye disease (GO) or to diagnose Graves' eye disease (GO) by comparing the measured concentration of lipid components with the concentration of lipid components isolated from the normal group. .

In the present invention, one or more selected from the group consisting of lysophosphatidylcholine (LPC) and triacylglycerol (TG) may be used as lipid components, that is, markers for identifying two diseases of the GD and GO. In this case, lysophosphatidylcholine (LPC) is at least one selected from the group consisting of 16: 1-LPC and 18: 1-LPC, triacylglycerol (TG) is 44: 2-TG, 54: 7-TG, 60:13 At least one selected from the group consisting of -TG, 46: 2-TG, 48: 1-TG, 50: 1-TG, 50: 2-TG, and 52: 0-TG.

In one embodiment, when the biological sample is blood, specifically plasma, the lipid component may be triacylglycerol (TG). At this time, the triacylglycerol (TG) may be one or more selected from the group consisting of 44: 2-TG, 54: 7-TG and 60: 13-TG.

In the blood sample, if two or more of the following conditions are satisfied, it may be determined as Graves' disease (GD). Graves 'disease (GD) at this time includes Graves' eye disease (GO).

(1) The concentration of 44: 2-TG is 1.5 times higher than that of the normal group

(2) 54: 7-TG concentration is 1.5 times more than that of normal group

(3) the concentration of 60: 13-TG is 1.5 times higher than that of the normal group

In addition, when two or more of the following conditions are satisfied in the blood sample, it may be determined as Graves 'eye disease (GO) which is distinguished from Graves' disease (GD).

(1) The concentration of 44: 2-TG is 2.5 times higher than that of the normal group

(2) 54: 7-TG concentration is 2.2 times more than that of normal group

(3) The concentration of 60: 13-TG is 2.5 times higher than that of the normal group

In other words, 44: 2-TG, 54: 7-TG, and 60: 13-TG in blood samples have a difference in concentration of 1.5 times or more compared to the normal group. It is used as a marker for. Furthermore, since the markers have a concentration difference of 30% or more in Graves 'disease (GD) and Graves' eye disease (GO) and also have the same increase / decrease tendency of Graves 'disease (GD) and Graves' eye disease ( GO) It serves as a marker for the identification of patients. Accordingly, the marker makes it possible to distinguish between a normal person, Graves' disease (GD) patient and Graves' eye disease (GO) patient, and further determine Graves' eye disease (GO) from the subject.

On the other hand, in one embodiment, when the biological sample is urine, the lipid component may be one or more selected from the group consisting of lysophosphatidylcholine (LPC) and triacylglycerol (TG). In this case, lysophosphatidylcholine (LPC) is at least one selected from the group consisting of 16: 1-LPC and 18: 1-LPC, triacylglycerol (TG) is 46: 2-TG, 48: 1-TG, 50: 1 At least one selected from the group consisting of -TG, 50: 2-TG, and 52: 0-TG.

In the urine sample, if two or more of the following conditions are satisfied, it can be determined as Graves' disease (GD). Graves 'disease (GD) at this time includes Graves' eye disease (GO).

(1) The concentration of 16: 1-LPC is 1.5 times higher than that of the normal group

(2) the concentration of 18: 1-LPC is 1.5 times or more than that of the normal group

(3) The concentration of 46: 2-TG is 0.67 times less than that of the normal group.

(4) The concentration of 48: 1-TG is 0.67 times less than that of the normal group.

(5) 50: 1-TG concentration is 0.67 times less than that of normal group

(6) The concentration of 50: 2-TG is 0.67 times lower than that of the normal group.

(7) 52: 0-TG concentration is 0.67 times less than that of normal group

In addition, in a urine sample, when two or more of the following conditions are satisfied, it can be judged as Graves ocular disease (GO).

(1) Concentration of 16: 1-LPC is 4.5 times higher than that of normal group

(2) 18: 1-LPC concentration is 5.8 times or more than that of normal group

(3) the concentration of 46: 2-TG is 0.55 times or less than that of the normal group

(4) the concentration of 48: 1-TG is 0.55 times less than that of the normal group

(5) The concentration of 50: 1-TG is 0.45 times less than that of the normal group

(6) 50: 2-TG concentration is 0.45 times or less than that of normal group

(7) 52: 0-TG concentration is 0.55 times less than that of normal group

That is, in the urine, 16: 1-LPC, 18: 1-LPC, 46: 2-TG, 48: 1-TG, 50: 1-TG, 50: 2-TG, and 52: 0-TG are compared with the normal group. It is used as a marker to distinguish between settler and Graves' disease (GD) patients because they have different concentration differences of 1.5 times or 0.67 times. Furthermore, since the markers have a concentration difference of 30% or more in Graves 'disease (GD) and Graves' eye disease (GO) and also have the same increase / decrease tendency of Graves 'disease (GD) and Graves' eye disease ( GO) It serves as a marker for the identification of patients. Accordingly, the marker makes it possible to distinguish between a normal person, Graves' disease (GD) patient and Graves' eye disease (GO) patient, and further determine Graves' eye disease (GO) from the subject group.

In addition, the present invention is a diagnostic for identifying Graves 'disease (GD) and Graves' eye disease (GO) comprising a quantification device for a marker comprising at least one selected from the group consisting of lysophosphatidylcholine (LPC) and triacylglycerol (TG) Relates to a kit.

The metering device may be an ultra-high performance liquid chromatography-electrospray ionization-mass spectrometer (nLC-ESI-MS / MS).

Hereinafter, the present invention will be described in more detail through examples according to the present invention and comparative examples not according to the present invention, but the scope of the present invention is not limited to the examples given below.

Example

1. Securing Geology

In the present invention, Folch modified with MTBE / CH ₃ OH extraction using methyl tert-butyl ether (MTBE), which is less toxic than chloroform, was used.

The extraction method uses a solvent in which MTBE and methanol are mixed, and there is an advantage that the organic solvent layer is in the upper layer because of the relatively low density of MTBE when the aqueous solution layer and the organic solvent layer are separated. In addition, the extraction method can be extracted with a relatively high polarity of lyso lipids and S1P to a high non-polar TG recovery.

MTBE, CH ₃ OH and H ₂ O used for the extraction were purchased from Avantor ™ Performance Materials (Center Valley, PA) by HPLC-grade.

Lipids were extracted using the modified strain extraction method by the method of FIG. 1.

Specifically, lipids were extracted in 100 μl for plasma samples and 1 ml for urine samples. First, 300 μl of CH 3 OH was added, followed by brief mixing. The sample was placed in an ice bath for 10 minutes. Thereafter, 1000 μl of MTBE was added and then vortexed for 1 hour, 250 μl of H ₂ O was added, and then vortexed for 10 minutes. After centrifugation at 1000 X g to separate the aqueous layer and the organic layer, the organic layer present in the upper layer was taken. After taking the organic layer, 300 μl of CH3OH was added to the aqueous layer, followed by vortexing for 10 minutes, centrifugation at 1000 X g, followed by taking the organic layer present in the upper layer, combined with the previously taken organic layer, and drying through a freeze dryer roll. I was. The dried lipid was dissolved in chloroform CHCl 3: CH 3 OH (3: 7, v / v) and analyzed by dilution in CH 3 OH: H 2 O (9: 1, v / v).

2. Measurement of Lipid Concentration Marker  Selection

Lipids extracted from 100 μl plasma and 1 mL urine samples were subjected to qualitative and quantitative analysis of lipids using the ultra-high performance liquid chromatography-electrospray ionization-mass spectrometer according to FIG. 2. Capillary with an inner diameter of 100 μm purchased from Polymicro Technology, LLC (Phoenix, AZ) was filled with ethylene bridged hybrid bead (BEH) from Waters (Milford, MA) having a size of 1.7 μm using nitrogen gas. Produced by direct packing (packing). The pump and column connections all used capillary with an internal diameter of 50 μm and a valve with an internal diameter of 20 μm for a valve that splits the flow rate for nanoflow. The split flow was controlled by connecting capillary.

Qualitative analysis was carried out by directly connecting ThermoFisher Scientific's Thermo Scientific ™ UltiMate ™ 3000 RSLCnano System to the LTQ velos ion trap mass spectrometer. NanoACQUITY UPLC was used in conjunction with a TSQ Vantage triple-stage quadrupole mass spectrometer.

The mobile phase used in the separation was separated in order from the highly polar material by using a gradient elution method. Mobile phase A uses a solvent in which H ₂ O and CH ₃ CN are mixed at a volume ratio of 9: 1, and mobile phase B has a ratio of 2: 2: 6 of CH ₃ OH: CH ₃ CN: isopropanol (IPA). A mobile phase mixed with was used. Mobile phase A is a highly polar solvent compared to B. When nonpolar lipid molecules are injected into the column through the autosampler, the mobile phase A effectively binds the column and the lipid molecules without falling out of the column by the mobile phase. Do it. When the sample is injected into the column, it moves only to the mobile phase A and proceeds with the split valve closed. The composition of mobile phase B increased the proportion of highly non-polar IPA, allowing efficient separation of nonpolar lipid molecules bound to the column. However, if the ratio of IPA is too high, the pressure generated when the mobile phases A and B are mixed increases, so that CH ₃ CN having the lowest viscosity is added to the mobile phase B that can be used. ₃ OH was also added. Slowly increase the proportion of mobile phase B to change the position of the split valve when separating the lipids so that only 300 nL / min of the flow rate from the pump flows to the analytical column, and the remaining flow rate flows out of the split valve. By moving out, the mobile phase solvent change was applied quickly and the dwell time was minimized.

Another gradient elution method was used that was optimized for qualitative and quantitative analysis. In the case of qualitative analysis (LTQ Velos ion trap), the sample was flowed at 600 nL / min for 13 minutes to sufficiently bind the analysis column. Then open the split valve and increase the flow rate to 6 μL / min, then move Mobile Phase B to 75% for 2 minutes, 80% for 9 minutes, 95% for the next 9 minutes, 99% for the next 9 minutes and 99% Was maintained for 10 minutes to separate and analyze lipids. For quantitative analysis (TSQ Vantage triple-quadrupole), flow mobile phase A at 1 μL / min for 10 minutes, bind the sample to the column, open split valve, and flow rate 20 μL / min for mobile phase B. Lipids were quantified in just 30 minutes at 50% for 1 minute, 80% for the next 3 minutes, 100% for the next 9 minutes, and 100% for 8 minutes.

Lipid analysis was performed using a mobile phase solvent containing 1 mM Ammonium formate (AF), 0.1% formic acid (FA), 1 mM AF and 1% FA, 5 mM AF, 1% FA, and 1 mM AF. As shown in Fig. 3, S1P was detected only when FA was added at 1% or more. In the case of AF, it was proved that the ionization efficiency increased when 1 mM lower than 5 mM was added, so the final selection of the ionization medium mixed with 1 mM AF and 1% FA was performed to analyze various types of lipids at once. It was. AF and FA were purchased from Sigma-Aldrich (St. Louis, Mo.) and all solvents used in the mobile phase composition were purchased from Avantor ™ Performance Materials (Center Valley, PA), respectively, by HPLC-grade.

The analysis was performed by injecting 5 ㎍ of plasma lipids and 15 ㎍ of urine sample into the normal, GD, and GO groups using ionization media and gradient elution optimized for lipid analysis.

In the case of qualitative analysis, the samples were pooled by the normal group, the GD group, and the GO group, and the analysis was performed. As shown in FIG. 4, the lipid structure was identified by using collision-induced dissociation (CID) spectra. It was.

In the case of S1P, in the cation mode, it was detected in [M + H] ⁺ form and detected by cutting off H ₂ O (m / z 362.4), by cutting off phosphate groups (m / z 282.4), and in the phosphate group H ₂ O was further cut and detected fragment ions (m / z 264.4) were detected to confirm the structure of S1P. In negative ion mode [MH] ^- is detected in the form of a phosphoric ion (m / z 97) and H ₂ O is added to the ion (m / z 79) is shown the structure analysis is possible in the negative ion mode, cut into, but the detected intensity of the cation mode Much higher relationships were analyzed in cationic mode for S1P. In this way, a total of 389 lipid structures were analyzed in blood samples and 273 in urine samples (Table 2).

Since it takes a lot of time to individually quantify hundreds of lipids from more than 150 samples, the first quantification was conducted with a pooled sample to reduce the analysis time. The quantitative analysis was performed in the Selective Reaction Monitoring (SRM) mode, where the precursor value and the product ion were selected to analyze only selective lipids. Urine lipids were 191 species (Table 2).

Classes Plamsa Urine Classes Plamsa Urine Classes Plamsa Urine LPC 16 7 LPI 2 One SM 23 21 PC 39 26 PI 20 16 CER 13 8 LPE 7 3 LPS 0 One MHC 9 7 PE 20 13 PS 0 10 DHC 4 5 PEp 13 7 PA 5 5 THC 4 4 LPG 6 3 S1P One One DG 23 10 PG 22 11 LSM 4 4 TG 50/158 28/110 TOTAL 281/389 191/273

In the case of TG, unlike the other lipids, the location of fatty acids is not discernible, and because they consist of three fatty acids, the same type of fatty acids are present in different combinations. For example, 46: 0-TG has 46 carbons and 14: 0, 16: 0, 18: 0 fatty acids were detected in the CID spectra, and 28: 0, 30: 0, 32: 0 was detected. It was analyzed that 46: 0-TG exists as 14: 0,16: 0,16: 0-TG and 14: 0,14: 0,18: 0-TG. So all TG isomers that have fatty acids of that length were analyzed together. For this reason, the TG detected in the quantitative analysis was relatively reduced compared to the number of TG detected in the qualitative analysis. In the first quantitative analysis, the analysis time was shortened by performing individual quantification of each lipid only sample that showed a difference of 1.5 times or more including the standard deviation.

Tables 3 to 5 show only the lipids (bold) showing a difference of 1.5 times or more and p <0.01 by comparing the normal group (C), the GD group, and the GO group in the individual sample quantitative analysis results. In addition, in FIG. 5, the peak area of each lipid was calculated and represented as GO / GD compared to the GD / C, GO / C and GD and GO groups compared to the normal group. In order to correct the ionization change, the peak area of the lipid internal standard added to each sample was calculated to correct the peak area of all lipids.

Lipids summarized in Tables 3 to 5 are depicted by principal component analysis (PCA) (FIG. 5).

Each point represents an individual sample. The normal group is represented by black circles, the GD group by white circles, and the GO group by stars. The fact that the grouping was clustered in each group showed that the distribution was good in the group, and it could be concluded that the GD group and the GO group did not overlap in the blood sample than the urine sample.

FIG. 6 is a diagram illustrating changes in specific lipid molecules of the GD group and the GO group with respect to the normal group.

A total of 5 types of lipids in plasma and 9 types of urine samples were found to be statistically significant (p <0.01) with more than 1.5-fold increase and decrease in the GD and GO groups.

Especially, d18: 1-S1P of GO group was found to increase statistically in plasma and urine samples, respectively. In the plasma samples, 16: 0p / 20: 2-PEp increased in the GO group but did not change significantly in the GD group, while 58: 7-TG increased in the GD group but did not change or decreased slightly in the GO group. d18: 1-S1P and (16: 1,18: 0) -DG increased in the GD and GO groups, but significantly increased in the GO group, indicating a statistically significant difference. In the urine sample, 16: 0/22: 5-PE, 18: 0/20: 4-PE, 18: 1/18: 2-PE, and 18: 1/20: 4-PE were compared with the normal group. The decrease in was greater than the decrease in the GO group, whereas for 48: 1- and 50: 1-TG, the decrease in the GO group was greater. 16: 1-LPC and d18: 1-S1P increased more rapidly in the GO group than in the GD group, and 16: 1-LPC increased about 3 times in the GD group and about 6 times in the GO group, resulting in other blood or urine samples. Notice the significant increase over any lipid.

Claims (10)

delete delete Measuring the concentration of lipid component in a biological sample isolated from the subject; And
Comparing the concentration of the lipid component and the concentration of the lipid component isolated from the normal group comprising the step of determining whether Graves 'disease (GD) or Graves' eye disease (GO),
The lipid component comprises at least one selected from the group consisting of lysophosphatidylcholine (LPC) and triacylglycerol (TG)
Method of providing information to identify Graves 'disease (GD) or Graves' eye disease (GO).
The method of claim 3, wherein
Determination of the concentration of lipid components using chromatography / mass spectrometry
Method of providing information to identify Graves 'disease (GD) or Graves' eye disease (GO).
The method of claim 3, wherein
Lysophosphatidylcholine (LPC) is at least one selected from the group consisting of 16: 1-LPC and 18: 1-LPC, triacylglycerol (TG) is 44: 2-TG, 54: 7-TG, 60: 13-TG At least one selected from the group consisting of: 46: 2-TG, 48: 1-TG, 50: 1-TG, 50: 2-TG, and 52: 0-TG.
Method of providing information to identify Graves 'disease (GD) or Graves' eye disease (GO).
The method of claim 3, wherein
Biological samples are tissue, blood or urine
Method of providing information to identify Graves 'disease (GD) or Graves' eye disease (GO).
The method of claim 6,
If the biological sample is blood, the lipid component is triacylglycerol (TG),
The triacylglycerol (TG) is at least one selected from the group consisting of 44: 2-TG, 54: 7-TG and 60: 13-TG
Method of providing information to identify Graves 'disease (GD) or Graves' eye disease (GO).
The method of claim 7, wherein
If two or more of the following conditions are satisfied, it is determined as Graves' disease (GD),
(1) The concentration of 44: 2-TG is 1.5 times higher than that of the normal group
(2) 54: 7-TG concentration is 1.5 times more than that of normal group
(3) the concentration of 60: 13-TG is 1.5 times higher than that of the normal group,
If two or more of the following conditions are met, Graves ocular disease (GO) is determined,
(1) The concentration of 44: 2-TG is 2.5 times higher than that of the normal group
(2) 54: 7-TG concentration is 2.2 times more than that of normal group
(3) the concentration of 60: 13-TG is 2.5 times higher than that of the normal group,
The Graves 'disease (GD) is to include Graves' eye disease (GO)
Method of providing information to identify Graves 'disease (GD) or Graves' eye disease (GO).
The method of claim 6,
If the biological sample is urine, the lipid component is at least one selected from the group consisting of lysophosphatidylcholine (LPC) and triacylglycerol (TG),
The lysophosphatidylcholine (LPC) is at least one selected from the group consisting of 16: 1-LPC and 18: 1-LPC, triacylglycerol (TG) is 46: 2-TG, 48: 1-TG, 50: 1- One or more selected from the group consisting of TG, 50: 2-TG and 52: 0-TG
Method of providing information to identify Graves 'disease (GD) or Graves' eye disease (GO).
The method of claim 9,
If two or more of the following conditions are satisfied, it is determined as Graves' disease (GD),
(1) The concentration of 16: 1-LPC is 1.5 times higher than that of the normal group
(2) the concentration of 18: 1-LPC is 1.5 times or more than that of the normal group
(3) The concentration of 46: 2-TG is 0.67 times less than that of the normal group.
(4) The concentration of 48: 1-TG is 0.67 times less than that of the normal group.
(5) 50: 1-TG concentration is 0.67 times less than that of normal group
(6) The concentration of 50: 2-TG is 0.67 times lower than that of the normal group.
(7) the concentration of 52: 0-TG is 0.67 times or less than that of the normal group,
If two or more of the following conditions are met, Graves ocular disease (GO) is determined,
(1) Concentration of 16: 1-LPC is 4.5 times higher than that of normal group
(2) 18: 1-LPC concentration is 5.8 times or more than that of normal group
(3) the concentration of 46: 2-TG is 0.55 times or less than that of the normal group
(4) the concentration of 48: 1-TG is 0.55 times less than that of the normal group
(5) The concentration of 50: 1-TG is 0.45 times less than that of the normal group
(6) 50: 2-TG concentration is 0.45 times or less than that of normal group
(7) the concentration of 52: 0-TG is 0.55 times or less than that of the normal group,
The Graves 'disease (GD) is to include Graves' eye disease (GO)
How to provide information to identify Graves 'disease (GD) or Graves' eye disease (GO):

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