Sulfur compound measurement method and disease determination method

Abstract

The present invention is a sulfur compound measurement method for diagnosing a disease, the method comprising a measurement step for using a mass spectrometer to analyze a sample obtained from the exhaled breath of a subject, and measuring the amounts of a plurality of types of sulfur compounds contained in the sample, the measurements being used for diagnosing whether the subject is suffering from a prescribed disease. The plurality of types of sulfur compounds include two or more types of sulfur compounds selected from among ten types of sulfur compounds, namely hydrogen sulfide, hydrogen disulfide, hydrogen trisulfide, hydrogen tetrasulfide, cysteine, cysteine persulfide, glutathione, glutathione persulfide, sulfurous acid, and thiosulfuric acid.

Description

Method for Measuring Sulfur Compound and Method for Determining Disease

[0001] The present invention relates to a method for measuring a sulfur compound contained in a sample (biological sample) collected from a subject and a method for determining a disease for determining whether the subject has a disease or not.

[0002] Irritable bowel syndrome, which is one of the digestive diseases, has symptoms such as abdominal abnormalities such as abdominal pain and abdominal discomfort, and bowel movement abnormalities such as constipation or diarrhea. It is a functional disease in which no lesions are observed in the organs of the digestive tract. Irritable bowel syndrome is thought to be caused by psychological factors such as mental stress and autonomic nerve disorders, but the exact cause has not been identified. Therefore, it is difficult to diagnose from the results of blood tests, gastrointestinal endoscopy, etc., and it is often diagnosed as irritable bowel syndrome only after excluding other digestive diseases. As a result, the start of treatment suitable for irritable bowel syndrome is delayed.

[0003] In addition, digestive cancers such as gastric cancer, colorectal cancer, and esophageal cancer all have a high cure rate if treatment is started at an early stage, and the physical burden caused by treatment is also small. However, since digestive cancers have almost no subjective symptoms at an early stage, there are many cases where they are left untreated without visiting a medical institution, and early detection and early treatment are difficult.

[0004] Under such circumstances, if it is possible to examine whether a person who visits a medical institution with the symptoms of irritable bowel syndrome has irritable bowel syndrome or digestive cancer, it will lead to early detection of irritable bowel syndrome and digestive cancer. In addition, if it is possible to find early-stage irritable bowel syndrome and early-stage digestive cancer through a simple test such as a general health check and start treatment, the cure rate of irritable bowel syndrome and digestive cancer can be increased.

[0005] In recent years, substances called reactive sulfur, which are present in large amounts in various organs and blood in the body, have attracted attention. Reactive sulfur is a general term for highly reactive sulfur compounds represented by cysteine persulfide in which an excessive amount (usually two or more) of sulfur atoms are added to the thiol (SH) group of cysteine, and it has been found to function as a major antioxidant that exhibits the ability to eliminate reactive oxygen in the body.

[0006] Japanese Patent Publication No. 2022-178150

[0007] Numakura et al. (2017) Production of reactive persulfide species in chronic obstructive pulmonary disease. Thorax, 72, pp1074-1083Kyogoku et al. (2019) Ni trosati ve stress in patients with asthma-chronic obstructive pulmonary disease overlap. J. Allergy Clin. Immunol., 144, pp972-983. e14.Fukuoka et al. (2021) Sulfur metabolism in colon cancer tissues: a case report and literature review. J. Int. Med. Res., (2021), 49(11), pp1-11

[0008] To date, it has been reported that active sulfur levels are reduced in lung cells and airway epithelial fluid obtained from patients with chronic obstructive pulmonary disease and related inflammatory airway diseases (Non-Patent Documents 1 and 2), and that levels of substances related to active sulfur are higher in colorectal cancer tissue than in normal tissue (Non-Patent Document 3). However, tissue biopsies are not suitable as a general health checkup item because they place a significant burden on the subject due to the need to collect tissue from within the body. Furthermore, while it has been reported that active sulfur contained in the subject's exhaled breath can be used as a biomarker for infectious disease diagnosis (Patent Document 1), this biomarker is targeted at respiratory diseases such as interstitial pneumonia caused by COVID-19 infection and other alveolar pneumonias, and it is unclear whether active sulfur contained in exhaled breath can serve as a biomarker in diseases other than respiratory diseases.

[0009] The problem that this invention aims to solve is to enable the determination of whether or not a subject is suffering from a predetermined disease by measuring substances contained in a sample non-invasively collected from the subject.

[0010] The present invention, made to solve the above problems, is a method for measuring sulfur compounds, which includes a measurement step of measuring the amount of multiple types of sulfur compounds contained in a sample obtained from the breath of a subject, which is used to determine whether or not the subject is suffering from a predetermined disease, and wherein the multiple types of sulfur compounds include two or more sulfur compounds selected from 10 types of sulfur compounds: hydrogen sulfide (HSH), hydrogen disulfide (HSSH), hydrogen trisulfide (HSSSH), hydrogen tetrasulfide (HSSSSH), cysteine ​​(CysSH), cysteine ​​persulfide (CysSSH, etc.), glutathione (GSH), glutathione persulfide (GSSH, etc.), sulfite (H2SO3), and thiosulfate (H2S2O3).

[0011] According to the sulfur compound measurement method of the present invention, it is possible to easily determine whether or not a subject is suffering from a predetermined disease based on the results of measuring two or more predetermined sulfur compounds contained in a sample non-invasively collected from the subject using a mass spectrometer.

[0012] A schematic diagram of an example of an analytical system for implementing a sulfur compound measurement method according to one embodiment of the present invention. A diagram showing a list of 10 sulfur compounds to be measured. A diagram showing a list of sulfur compounds other than the above 10 that may be measured. A diagram showing an example of derivatization to stabilize active sulfur. A graph showing the concentrations of 4 sulfur compounds (HSH, HSSH, HSSSH, HSSSSH) contained in exhaled breath coagulation (EBC samples) obtained from esophageal cancer patients and healthy individuals (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001). A graph showing the concentrations of 7 sulfur compounds contained in EBC samples obtained from patients with 5 diseases and healthy individuals. A diagram showing the results of multivariate analysis integrating the concentrations of 10 sulfur compounds contained in EBC samples obtained from patients with 6 diseases and healthy individuals.

[0013] Hereinafter, a sulfur compound measurement method, which is one embodiment of the present invention, will be described with reference to the attached drawings.

[0014] [Sulfur compounds to be measured] Figure 2 shows the 10 sulfur compounds to be measured in the measurement method of this embodiment: hydrogen sulfide (HSH), hydrogen disulfide (HSSH), hydrogen trisulfide (HSSSH), hydrogen tetrasulfide (HSSSSH), cysteine ​​(CysSH), cysteine ​​hydrodisulfide (CysSSH), glutathione (GSH), glutathione hydrodisulfide (GSSH), sulfite (H2SO3), and thiosulfate (H2S2O3).

[0015] As shown in the right column of Figure 2, the 10 types of sulfur compounds can be divided into four groups. In these four groups, the sulfur compounds marked with an asterisk in Figure 2 are chemically stable sulfur compounds and are not active sulfur in the strict sense. The other sulfur compounds that are not marked with an asterisk are chemically unstable and are also called active sulfur. The active sulfur compounds in the hydrogen sulfide group, cysteine ​​group, and reduced glutathione group have a highly reactive thiol group (-SH) and are particularly unstable active sulfur compounds with poor chemical stability.

[0016] Furthermore, while sulfur compounds in the cysteine ​​group and the reduced glutathione group are organic compounds, those in the hydrogen sulfide group and the sulfate group are inorganic compounds. Generally, although sulfur compounds that may be included in the sulfate group are not active sulfur compounds, they are important sulfur sources in metabolism within the body and can therefore be considered as sulfur compounds similar to active sulfur compounds.

[0017] As can be seen from Figure 2, in the four groups mentioned above, multiple sulfur compounds belonging to the same group are compounds in which only the number of sulfur atoms constituting the chain bond of sulfur atoms (...-S-S-...) differs, and the structure (composition) of the rest of the compound is identical. For example, the four sulfur compounds belonging to the hydrogen sulfide group have 1 to 4 sulfur atoms in the chain bond. There are also compounds in the hydrogen sulfide group that have 5 or more sulfur atoms, so such compounds can also be added to the same group. The same applies to the other groups. Furthermore, even if the structure other than the chain bond of sulfur atoms is not completely identical, they can still be included in the same group in some cases.

[0018] Furthermore, sulfur compounds other than the four groups mentioned above can also be included as targets for measurement. Figure 3 shows examples of sulfur compounds other than the four groups mentioned above that can be included as targets for measurement in this embodiment. The sulfur compounds shown in Figure 3 consist of the cystine group, the oxidized glutathione group, and the homocysteine ​​group, as shown in the right column of the figure. Similar to Figure 2, the sulfur compounds marked with an asterisk in Figure 3 are chemically stable sulfur compounds and are not active sulfur in the narrow sense. On the other hand, the sulfur compounds that are not marked with an asterisk are chemically unstable and are also called active sulfur.

[0019] As can be seen from Figures 2 and 3, sulfur compounds in which the number of sulfur atoms constituting the chain bond is one or more are sulfur compounds that can be included in the measurement target.

[0020] [Pretreatment for stabilization of activated sulfur] The unstable activated sulfur described above is difficult to analyze directly using a mass spectrometer. Therefore, pretreatment by derivatization is performed to stabilize the unstable activated sulfur. Specifically, β-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM) is used as the derivatization reagent.

[0021] Figure 4 shows the derivatization reaction using HPE-IAM. HPE-IAM selectively reacts with the SH group contained in reduced active sulfur, derivatizing the active sulfur. The OH group contained in the derivative suppresses side reactions to the polysulfide chain, making it chemically stable. In this specification, when the abbreviation of a compound name includes "-HPE", it indicates that the derivative has been derivatized by HPE-IAM. Although such derivatization is substantially essential as a pretreatment for measurement of reduced active sulfur, the use of HPE-IAM as a reagent is not essential.

[0022] [Sample for Measurement] In the measurement method of this embodiment, the amount of sulfur compounds in the breath collected from the subject is measured as a biomarker for determining whether or not the subject is suffering from a predetermined disease. Here, "subject" is typically a human, but may also be a primate or a mammal.

[0023] Furthermore, the "specified diseases" may include a variety of diseases such as gastrointestinal cancer, irritable bowel syndrome, breast cancer, COVID-19, and mental illnesses such as depression and bipolar disorder. The measurement method of this embodiment is suitable for measuring sulfur compounds that are useful as biomarkers for determining whether a subject is suffering from a disease in which symptoms are not visible or are difficult to notice in the early stages. Therefore, the "specified diseases" are suitable to be diseases in which symptoms are not visible or are difficult to notice in the early stages.

[0024] Gastrointestinal cancers are cancers that occur in the digestive system, and in their early stages, there are usually no noticeable symptoms. In this respect, gastrointestinal cancers are suitable as a "specified disease." The digestive system refers to the organs responsible for the intake, absorption, digestion, transport, absorption of digested food, and excretion of food, and includes, for example, the oral cavity, pharynx, esophagus, stomach, small intestine, large intestine, liver, gallbladder, pancreas, and peritoneum, or parts of these or their appendages. Examples of "gastrointestinal cancers" include esophageal cancer, stomach cancer, colon cancer, small intestine cancer, liver cancer, gallbladder cancer, pancreatic cancer, and bile duct cancer.

[0025] Gastrointestinal cancers include squamous cell carcinoma and adenocarcinoma. Squamous cell carcinoma is cancer that originates from squamous epithelium, while adenocarcinoma is cancer that originates from glandular cells (glandular epithelial cells). Breast cancer, on the other hand, refers to cancer that originates from the ducts and lobules of the mammary gland tissue. Gastrointestinal cancers and breast cancers can be at any stage (clinical stage), including clinical stages I-II and more advanced stages III-IV.

[0026] Irritable bowel syndrome (IBS) presents with symptoms such as abdominal pain and bowel irregularities, but no organic lesions are found, leading to the suggestion that it may be caused by psychological factors such as mental stress or autonomic nervous system dysfunction. Similarly, mental illnesses such as depression and bipolar disorder are thought to be partly caused by excessive psychological or physical stress. Many diseases caused by psychological factors do not involve organic lesions, making them difficult to diagnose with tests such as blood tests or endoscopy. The present invention is suitable for measuring sulfur compounds used as biomarkers to determine whether or not a subject suffers from such a disease; therefore, diseases caused by psychological factors such as irritable bowel syndrome, depression, and bipolar disorder are suitable as "specified diseases" in the present invention.

[0027] The sample used in this measurement method may be the exhaled breath itself collected from the subject, or a sample prepared from the exhaled breath collected from the subject. Any sample containing exhaled breath is acceptable, but if the sulfur compound to be measured contains both volatile and non-volatile compounds, it is preferable to use exhaled breath condensates (EBC). Exhaled breath condensates are liquids obtained by cooling and condensing exhaled breath at a low temperature (for example, -20°C to 0°C).

[0028] Exhaled breath condensate can be obtained by collecting it from the subject using a mouthpiece or similar device and then cooling it, but it is preferable to obtain it using the exhaled breath condensate collection device of GL Sciences Inc. Other options include ECoScreen (registered trademark, JAEGER GmbH (Germany)), Turbo DECCS system (Medivac GmbH (Italy)), and RTube. TM Commercially available breath condensate collection devices, such as those from Resptratory Research (USA), may also be used.

[0029] [Measurement of Active Sulfur] In the measurement method of this embodiment, a mass spectrometer is used to comprehensively measure multiple types of sulfur compounds contained in the sample for measurement. Among mass spectrometers, a chromatograph mass spectrometer is particularly preferred, and if the sample for measurement is exhaled breath condensate, a liquid chromatograph mass spectrometer (LC-MS) is even more preferred.

[0030] When a liquid chromatography-tandem mass spectrometer (LC-MS / MS) is used to measure a sample, the LC-MS / MS system works as follows: the liquid chromatograph separates multiple types of sulfur compounds and various other impurities in the sample over time, and the tandem mass spectrometer selectively detects each of the multiple types of sulfur compounds, obtaining ionic intensity signals corresponding to their amounts.

[0031] In a tandem mass spectrometer, MRM (Multiple Reaction Monitoring) measurements are performed targeting multiple reaction transitions, which are pairs of specific precursor and product ion mass-charge ratios (m / z) corresponding to each sulfur compound, within a predetermined measurement time range near the retention time corresponding to each of the multiple sulfur compounds. This provides data that constitutes an MRM chromatogram (hereinafter simply referred to as a chromatogram) for each of the multiple sulfur compounds. If a certain sulfur compound is present in the sample for measurement, a peak will appear in the chromatogram corresponding to that sulfur compound. Since the area (or height) of this peak depends on the amount or concentration of that sulfur compound, quantitative values ​​such as content and concentration can be determined based on the area or height value.

[0032] [Example of Analysis System Configuration] Figure 1 shows an example of an analysis system used for measuring sulfur compounds contained in a sample for measurement. This analysis system is an LC-MS / MS system that includes a measurement unit containing a liquid chromatograph (LC) 1 and a mass spectrometer 2, a data processing unit 3, an analysis control unit 4, a central control unit 5, an input unit 6, and a display unit 7.

[0033] The liquid chromatograph 1 includes a mobile phase container 11 in which the mobile phase (solvent) is stored, a liquid delivery pump 12 for aspirating and supplying the mobile phase from the mobile phase container 11, an injector 13 for injecting the sample into the mobile phase, and a column 14 for separating multiple components contained in the sample in the time direction. Although not shown in the figure, an autosampler is usually connected to the injector 13 to sequentially analyze a large number of samples.

[0034] The mass spectrometer 2 shown in Figure 1 is a triple quadrupole mass spectrometer, a type of tandem mass spectrometer, and comprises an ionization chamber 201 maintained at approximately atmospheric pressure, and a first intermediate vacuum chamber 202, a second intermediate vacuum chamber 203, and a high vacuum chamber 204, each evacuated by a vacuum pump (not shown). The ionization chamber 201 is equipped with an ESI spray 21 for ionization by the electrospray ionization (ESI) method, and the ionization chamber 201 and the next stage, the first intermediate vacuum chamber 202, are connected by a desolvation tube 22. An ion guide 23 for transporting ions while focusing them is located in the first intermediate vacuum chamber 202, and the first intermediate vacuum chamber 202 and the next stage, the second intermediate vacuum chamber 203, are connected through a small hole formed at the top of a skimmer 24. A multipole type ion guide 25 for transporting ions while focusing them is also located in the second intermediate vacuum chamber 203.

[0035] Inside the high vacuum chamber 204, a pre-stage quadrupole mass filter 26, a collision cell 27, a post-stage quadrupole mass filter 28, and an ion detector 29 are arranged along the flow of ions. A quadrupole-type ion guide is placed inside the collision cell 27. The pre-stage quadrupole mass filter 26 and the post-stage quadrupole mass filter 28 each have the function of selectively passing ions having a predetermined m / z. An inert collision-induced dissociation (CID) gas such as argon is introduced into the collision cell 27 from the outside, and the introduced ions are dissociated by contacting them with the CID gas to generate product ions.

[0036] The data processing unit 3 receives detection data from the ion detector 29 and performs processing based on that data. Its functional blocks include a data acquisition unit 31, a quantitative calculation unit 32, and a quantitative reference information storage unit 33. The analysis control unit 4 controls the operation of the liquid chromatograph 1 and the mass spectrometer 2 according to a sulfur compound analysis method (method file) 41, which contains information indicating analysis conditions specifically for the quantitative determination of sulfur metabolites and is stored in an internal storage unit. The central control unit 5 primarily performs overall control of each unit and provides a user interface through the input unit 6 and the display unit 7. As described above, the sulfur compound analysis method 41 includes individual MS analysis conditions for each sulfur compound.

[0037] Generally, the data processing unit 3, the analysis control unit 4, and the central control unit 5 are actually personal computers or more powerful workstations, and the functions of each of these functional blocks can be realized by running dedicated software (computer programs) pre-installed on these computers. In other words, the sulfur compound analysis method 41 is also a type of program that provides parameters for analysis and procedures for data processing.

[0038] Next, the present invention will be described more specifically with reference to examples, but these examples are merely illustrative and do not limit the scope of the present invention in any way.

[0039] [Subjects] The subjects were patients and healthy individuals who visited Tohoku University Hospital between June 2021 and September 2021. Healthy individuals were recruited and accepted on the condition that they had no history of cancer or surgery. Exhaled blood condensate (EBC) samples were collected before the commencement of any treatment, including drug therapy, chemotherapy, radiation therapy, surgery, endoscopic treatment, and dental treatment. Subjects were not restricted in terms of diet, smoking, alcohol consumption, exercise, etc. Written informed consent was obtained from all subjects. The procedures for this study were approved by the Ethics Committee of the Graduate School of Medicine (accession no. 2023-1-800).

[0040] [Collection of EBC] EBC was collected using a non-invasive device consisting of a Peltier device (Gel Science Co., Ltd.), a mouthpiece, and a 50 mL polystyrene tube. In the non-invasive device, the polystyrene tube was installed inside the Peltier device, and the mouthpiece was connected to the polystyrene tube.

[0041] The seated subject was fitted with a nose clip and instructed to breathe from the mouthpiece at a normal breathing frequency. Then, the subject was asked to breathe for 5 - 10 minutes with the temperature inside the Peltier device set to -20°C. As a result, the exhaled breath flowing from the mouthpiece into the polystyrene tube was rapidly frozen and condensed at a temperature of -20°C, and 0.5 - 1.0 mL of EBC was obtained. The EBC was stored at -80°C until the analysis was completed.

[0042] [Measurement of sulfur compounds] The mass spectrometer, reagents, and the content of the statistical processing of the obtained results used for the measurement of sulfur compounds contained in EBC are as follows. 1. Mass spectrometer High-performance liquid chromatography triple quadrupole mass spectrometer (Product name: LCMS - 8060NX, Shimadzu Corporation)

[0043] 2. Measurement conditions Column: YMC - Triart C18(2.0×50) (Waters Corporation) Mobile phase A: 0.1% formic acid Mobile phase B: Methanol containing 0.1% formic acid Flow rate: 0.2 mL / min Gradient: 0 - 3 min: 3%B, 3 - 15 min: 3 - 95%B, 15 - 16 min: 95%B, 16 - 16.1 min: 95 - 3%B, 16.1 - 19 min: 3%B ESI probe temperature: 300°C Desolvation line temperature: 250°C Heat block temperature: 400°C Nebulizer gas: 3 L / min Heating gas: 10 L / min Dry nitrogen gas: 10 L / min

[0044] 3. Reagents: - Derivatization reagent: s-4-hydroxyphenyl ethyl iodoacetamide (HPE-IAM) - Internal standard substance: Stable isotope-labeled HPE-IAM adduct internal standard (CysSH, CysSSH, CysSSSH, GSH, GSSH, GSSSH, HomoCysSH, HomoCysSSH, HS - , HSS - , HSSS - , HS03 - , HS203 - ) - Solvent: Dimethyl sulfoxide (DMSO)

[0045] 4. Pretreatment of EBC 50 μL of EBC and 4.0 μL of 5 mM HPE-IAM / DMSO were mixed and reacted at 37°C for 20 minutes (alkylation reaction). 0.1% formic acid containing a known amount of the internal standard substance was added to the reaction mixture to stop the alkylation reaction. The mixture after alkylation is hereinafter referred to as the EBC sample. 50 μL of the EBC sample was introduced into the mass spectrometer (LC-MS / MS) described above.

[0046] 5. Statistical analysis The measurement results of the EBC samples obtained by LC-MS / MS were expressed as "mean ± SD". Also, the measurement results of the EBC samples obtained by LC-MS / MS were processed using the statistical analysis software "GraphPad Prism version 9 (GraphPad Software Inc.)". Student's t-test was used for comparison between two groups, and one-way analysis of variance was used for comparison of three or more groups. Statistical significance was set at P < 0.05. To evaluate the value of sulfur compounds (metabolites) as biomarkers for a given disease, the ROC (Receiver Operating Characteristic) curve and the area under the curve (AUC) were calculated.

[0047] 6. Others Since the stability of other sulfur compounds is affected by many factors and the concentration may decrease, EBC samples containing EBC that has been repeatedly frozen and thawed, and EBC samples in which almost all sulfur compounds were not detected were excluded from the analysis target.

[0048] [Experiment 1] The amounts of hydrogen sulfide group sulfur compounds (HSH, HSSH, HSSSH, and HSSSSH) in EBC samples obtained from breast cancer patients (n=41) and healthy individuals (n=20) were measured. Figure 5 is a graph showing the measurement results. In the bar graph of Figure 5, the right side shows the measured values ​​(content) for breast cancer patients, and the left side shows the measured values ​​(content) for healthy individuals. As can be seen from Figure 5, there was no difference in the measured values ​​of hydrogen sulfide and hydrogen disulfide between breast cancer patients and healthy individuals among the four types of sulfur compounds, but there was a significant difference in the measured values ​​of hydrogen trisulfide and hydrogen tetrasulfide between breast cancer patients and healthy individuals, and the EBC levels of breast cancer patients were clearly higher than those of healthy individuals. In particular, considering that the measured value of hydrogen trisulfide was about 10 times that of hydrogen tetrasulfide, it was inferred that hydrogen trisulfide could be an effective biomarker for determining whether or not a subject has breast cancer.

[0049] [Experiment 2] The amounts of sulfur compounds from the hydrogen sulfide group (HSH, HSSH, HSSSH, HSSSSH), the cysteine ​​group (CysSH, CysSSH), and the reduced glutathione group (GSH, GSSH) were measured in EBC samples obtained from patients with five different diseases: depression, irritable bowel syndrome (IBS), COVID-19 infection, breast cancer, and esophageal cancer (depression patients (n=64), IBS patients (n=129), COVID-19 infection patients (n=22), breast cancer patients (n=41), and esophageal cancer patients (n=20)), as well as from healthy individuals (n=20). Figure 6 is a graph showing the measurement results. In the bar graph in Figure 6, the right side shows the measured values ​​(content) for patients, and the left side shows the measured values ​​(content) for healthy individuals.

[0050] The results showed that patients with depression had a significant increase in the inorganic sulfur compound HSH compared to healthy individuals, while organic sulfur compounds CysSH and CysSSH decreased. Patients with IBS showed an overall increase in both inorganic and organic sulfur compounds, including HSSH and HSSSH, and organic sulfur compounds CysSSH and GSSH. Patients with COVID-19 showed an increase in the inorganic sulfur compounds HSH and HSSH, but a decrease in HSSSH (another inorganic sulfur compound) and the organic compounds CysSSH and GSH. Breast cancer patients showed an increase in the inorganic sulfur compounds HSSH and HSSSH, while organic sulfur compounds decreased. Esophageal cancer patients showed an overall increase in organic sulfur compounds. These results clearly indicate that the metabolic dynamics profile of sulfur compounds differs depending on the disease.

[0051] [Experiment 3] The amounts of metabolites of 10 sulfur compounds (HSH, HSSH, HSSSH, HSSSSH, CysSH, CysSSH, GSH, GSSH, sulfite (H2SO3), thiosulfite (H2S2O3)) contained in EBC samples obtained from patients with the six diseases described above plus bipolar disorder, as well as from healthy individuals, were measured using a mass spectrometer. These measurements were then integrated and analyzed using UMAP (Uniform Manifold Approximation and Projection), a type of multivariate analysis. The results are shown in Figure 7. As can be seen from Figure 7, patients with the six diseases and healthy individuals formed distinct groups, each exhibiting disease-specific metabolic dynamics profiles of sulfur compounds.

[0052] From these results, it was inferred that by measuring the amounts of 10 types of sulfur compounds (sulfur metabolites) contained in EBC samples obtained from subjects using a mass spectrometer, and examining whether the results belong to one of the six disease groups or a healthy control group, it is possible to determine whether the subject suffers from one of the six diseases or none of them. In other words, it was shown that the 10 types of sulfur compounds (sulfur metabolites) can function as biomarkers used to determine whether or not a subject suffers from any of the various diseases. To put it another way, it was shown that by measuring the amount of sulfur compounds (sulfur metabolites) in a sample containing breath obtained from a subject using a mass spectrometer, it may be possible to determine whether or not the subject suffers from a predetermined disease.

[0053] One reason why, in this embodiment, it was possible to search for sulfur compounds that could serve as biomarkers for determining the six types of diseases from among the sulfur compounds contained in the subject's exhaled breath is that EBC was used to measure the sulfur compounds. In other words, with EBC, regardless of whether the sulfur compounds contained in the exhaled breath are volatile or non-volatile, the sulfur compounds in the EBC can be accurately analyzed using a mass spectrometer, and based on the results of that analysis, the patterns and correlations of the amounts of the ten types of sulfur compounds for each of the six types of diseases can be investigated.

[0054] Based on the above, the present invention is also directed to a disease determination method, which includes a sulfur compound measurement step of analyzing samples obtained from the breath of multiple patients suffering from one of six diseases: breast cancer, gastrointestinal cancer, irritable bowel syndrome, COVID-19, depression, and bipolar disorder, using a mass spectrometer, and measuring the amount of multiple types of sulfur compounds contained in the samples; a step of obtaining information related to the six diseases by performing multivariate analysis on the amount of multiple types of sulfur compounds contained in the samples obtained from the breath of the multiple patients, as measured in the sulfur compound measurement step; and a step of determining whether or not the subject suffers from one of the six diseases based on the results of analyzing a sample obtained from the breath of a subject using a mass spectrometer and measuring the amount of multiple types of sulfur compounds contained in the sample, and the information obtained above. The aforementioned multiple types of sulfur compounds include at least 10 types of sulfur compounds, namely hydrogen sulfide (HSH), hydrogen disulfide (HSSH), hydrogen trisulfide (HSSSH), hydrogen tetrasulfide (HSSSSH), cysteine ​​(CysSH), cysteine ​​persulfide (CysSSH), glutathione (GSH), glutathione persulfide (GSSH), sulfite (H2SO3), and thiosulfate (H2S2O3).

[0055] [Embodiments] It will be apparent to those skilled in the art that the exemplary embodiments described above are specific examples of the following embodiments.

[0056] (Section 1) One aspect of the present invention is a method for measuring sulfur compounds, which includes a measurement step of measuring the amount of a plurality of sulfur compounds contained in a sample obtained from the breath of a subject, for use in determining whether or not the subject is suffering from a predetermined disease, wherein the plurality of sulfur compounds include two or more sulfur compounds selected from 10 types of sulfur compounds: hydrogen sulfide (HSH), hydrogen disulfide (HSSH), hydrogen trisulfide (HSSSH), hydrogen tetrasulfide (HSSSSH), cysteine ​​(CysSH), cysteine ​​persulfide (CysSSH, etc.), glutathione (GSH), glutathione persulfide (GSSH, etc.), sulfite (H2SO3), and thiosulfate (H2S2O3).

[0057] In the present invention, cysteine ​​persulfide includes CysSSH (cysteine ​​hydrodisulfide), CysSSSH (cysteine ​​hydrotrisulfide), etc., in which the number of sulfur atoms constituting the chain of sulfur atoms (...-S-S-...) is two or more. Similarly, glutathione persulfide includes GSSH (glutathione hydrodisulfide), GSSSH (glutathione hydrotrisulfide), etc., in which the number of sulfur atoms constituting the chain of sulfur atoms (...-S-S-...) is two or more.

[0058] According to the sulfur compound measurement method described in paragraph 1, since exhaled breath collected non-invasively from the subject is used, the burden on the subject from taking samples for disease diagnosis can be reduced. Therefore, even for subjects who do not show symptoms of a specified disease, it is easy to obtain a sample from the subject to determine whether or not they have the disease, and by simply measuring two or more specified sulfur compounds contained in the sample using a mass spectrometer, it is possible to easily determine from the results whether or not the subject has the specified disease.

[0059] (Paragraph 2) The sulfur compound measurement method according to Paragraph 2 is the sulfur compound measurement method according to Paragraph 1, wherein the predetermined disease includes a plurality of predetermined diseases, and in the measurement step, the amount of a plurality of types of sulfur compounds contained in the sample is measured, which is used to determine whether or not the subject suffers from any of the plurality of predetermined diseases.

[0060] According to the sulfur compound measurement method described in paragraph 2, it is possible to determine whether or not a subject is suffering from one of several predetermined diseases at once by mass spectrometry analysis of a sample including exhaled breath obtained from the subject, thereby reducing the burden on the subject.

[0061] (Paragraph 3) The sulfur compound measurement method relating to Paragraph 3 is the sulfur compound measurement method relating to Paragraph 1 or Paragraph 2, wherein the mass spectrometer is a chromatograph-mass spectrometer.

[0062] According to the sulfur compound measurement method described in paragraph 3, multiple types of sulfur compounds contained in a sample, including the breath of a test subject, can be measured simultaneously.

[0063] (Article 4) The sulfur compound measurement method according to Article 4 includes a step of performing a pretreatment to derivatize the active sulfur contained in the sample using a predetermined derivatization reagent, wherein the measurement step measures the amount of multiple types of sulfur compounds (sulfur metabolites) contained in the sample that has undergone the pretreatment.

[0064] According to the sulfur compound measurement method described in paragraph 4, even chemically unstable sulfur compounds can be measured.

[0065] (Paragraph 5) The sulfur compound measurement method according to Paragraph 5 is the sulfur compound measurement method according to Paragraph 4, wherein the predetermined derivatization reagent is an electrophilic alkylating agent having a hydroxyphenyl group. The electrophilic alkylating agent having a hydroxyphenyl group selectively reacts with the SH group contained in the reduced active sulfur to derivatize the active sulfur. According to the sulfur compound measurement method according to Paragraph 5, the action of the OH group contained in the derivative suppresses side reactions to the polysulfide chain, so that the active sulfur becomes chemically stable and can be measured with high accuracy.

[0066] (Paragraph 6) The sulfur compound measurement method according to Paragraph 6 is the sulfur compound measurement method according to Paragraph 5, wherein the electrophilic alkylating agent having a hydroxyphenyl group is β-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM).

[0067] (Paragraph 7) The sulfur compound measurement method according to Paragraph 7 is the sulfur compound measurement method according to any one of Paragraphs 1 to 6, wherein the sample is a breath condensate obtained by cooling the breath of the subject.

[0068] According to the sulfur compound measurement method described in paragraph 7, sulfur compounds contained in the subject's exhaled breath can be accurately measured using a mass spectrometer, regardless of whether they are volatile or non-volatile. Therefore, the accuracy of determining whether a subject is suffering from one of the six diseases or none of the six diseases can be improved based on the results of the mass spectrometry analysis.

[0069] (Paragraph 8) The sulfur compound measurement method relating to Paragraph 8 is the sulfur compound measurement method relating to any one of Paragraphs 1 to 7, wherein the specified disease is one or more selected from breast cancer, gastrointestinal cancer, irritable bowel syndrome, and COVID-19.

[0070] (Paragraph 9) The sulfur compound measurement method relating to Paragraph 9 is the sulfur compound measurement method relating to any one of Paragraphs 1 to 7, wherein the specified disease is breast cancer and the multiple types of sulfur compounds are hydrogen trisulfide and hydrogen tetrasulfide.

[0071] (Paragraph 10) The sulfur compound measurement method relating to Paragraph 10 is the sulfur compound measurement method relating to any one of Paragraphs 1 to 7, wherein the specified disease is gastrointestinal cancer and the multiple types of sulfur compounds are cysteine ​​and cysteine ​​persulfide.

[0072] (Paragraph 11) The sulfur compound measurement method relating to Paragraph 11 is the sulfur compound measurement method relating to any one of Paragraphs 1 to 7, wherein the specified disease is irritable bowel syndrome, and the multiple types of sulfur compounds are two or more sulfur compounds selected from hydrogen disulfide, hydrogen trisulfide, cysteine ​​persulfide, and glutathione persulfide.

[0073] (Paragraph 12) The sulfur compound measurement method relating to Paragraph 12 is the sulfur compound measurement method relating to any one of Paragraphs 1 to 7, wherein the specified disease is COVID-19, and the multiple types of sulfur compounds are two or more sulfur compounds selected from hydrogen sulfide, hydrogen disulfide, hydrogen trisulfide, cysteine ​​persulfide, and glutathione.

[0074] (Paragraph 13) The sulfur compound measurement method relating to Paragraph 13 is the sulfur compound measurement method relating to any one of Paragraphs 1 to 7, wherein the specified disease is a mental illness, namely depression and bipolar disorder.

[0075] (Paragraph 14) The sulfur compound measurement method relating to Paragraph 14 is the sulfur compound measurement method relating to any one of Paragraphs 1 to 7, wherein the specified disease is depression, and the multiple types of sulfur compounds are two or more sulfur compounds selected from hydrogen trisulfide, cysteine, and cysteine ​​persulfide.

[0076] (Section 15) Another aspect of the present invention includes: a sulfur compound measurement step of analyzing samples obtained from the breath of multiple patients suffering from any of six diseases, namely breast cancer, gastrointestinal cancer, irritable bowel syndrome, COVID-19, depression, and bipolar disorder, using a mass spectrometer to measure the amount of multiple types of sulfur compounds contained in the samples; an information acquisition step of obtaining information related to the six diseases by performing multivariate analysis on the amounts of multiple types of sulfur compounds contained in the samples obtained from the breath of the multiple patients as measured in the sulfur compound measurement step; and a determination step of determining whether or not the subject suffers from any of the six diseases, based on the results of analyzing a sample obtained from the breath of a subject using a mass spectrometer to measure the amount of multiple types of sulfur compounds contained in the sample and the information. The aforementioned method for determining a disease includes two or more sulfur compounds selected from the following 10 sulfur compounds: hydrogen sulfide (HSH), hydrogen disulfide (HSSH), hydrogen trisulfide (HSSSH), hydrogen tetrasulfide (HSSSSH), cysteine ​​(CysSH), cysteine ​​persulfide (CysSSH, etc.), glutathione (GSH), glutathione persulfide (GSSH, etc.), sulfite (H2SO3), and thiosulfate (H2S2O3).

[0077] The information related to the six types of diseases refers, for example, to information that groups the patterns and correlations of the amounts of the ten types of sulfur compounds contained in a sample obtained from breath, according to the disease. Then, in the determination step, by examining which of the six types of diseases the patterns and interrelationships of the amounts of the ten types of sulfur compounds contained in the sample obtained from the subject's breath belong to, it is determined whether the subject is suffering from one of the six types of diseases or none of them.

[0078] 1. Liquid chromatograph 2. Mass spectrometer 3. Data processing unit 4. Analysis control unit 5. Central control unit 6. Input unit 7. Display unit

Claims

1. A method for measuring sulfur compounds, comprising a measurement step of measuring the amount of multiple types of sulfur compounds contained in a sample obtained from the breath of a subject, which is used to determine whether or not the subject is suffering from a predetermined disease, wherein the multiple types of sulfur compounds include two or more sulfur compounds selected from 10 types of sulfur compounds: hydrogen sulfide, hydrogen disulfide, hydrogen trisulfide, hydrogen tetrasulfide, cysteine, cysteine ​​persulfide, glutathione, glutathione persulfide, sulfite, and thiosulfate.

2. A method for measuring sulfur compounds according to claim 1, wherein the predetermined disease includes a plurality of predetermined diseases, and the amount of a plurality of types of sulfur compounds contained in the sample is measured in the measurement step, which is used to determine whether or not the subject suffers from any of the plurality of predetermined diseases.

3. The method for measuring sulfur compounds according to claim 1, wherein the mass spectrometer is a chromatograph-mass spectrometer.

4. A method for measuring sulfur compounds according to claim 1, comprising a step of performing a pretreatment using a predetermined derivatization reagent to derivatize the active sulfur contained in the sample, wherein the measurement step measures the amount of a plurality of types of sulfur compounds contained in the pretreated sample.

5. The method for measuring sulfur compounds according to claim 4, wherein the predetermined derivatization reagent is an electrophilic alkylating agent having a hydroxyphenyl group.

6. The method for measuring sulfur compounds according to claim 5, wherein the electrophilic alkylating agent having a hydroxyphenyl group is β-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM).

7. The method for measuring sulfur compounds according to claim 1, wherein the sample is a breath condensate obtained by cooling the breath of the subject.

8. The sulfur compound measurement method according to claim 1, wherein the predetermined disease is one or more selected from breast cancer, gastrointestinal cancer, irritable bowel syndrome, and COVID-19.

9. The method for measuring sulfur compounds according to claim 1, wherein the predetermined disease is breast cancer, and the plurality of sulfur compounds are hydrogen trisulfide and hydrogen tetrasulfide.

10. The method for measuring sulfur compounds according to claim 1, wherein the predetermined disease is gastrointestinal cancer, and the plurality of sulfur compounds are cysteine ​​and cysteine ​​persulfide.

11. The method for measuring sulfur compounds according to claim 1, wherein the predetermined disease is irritable bowel syndrome, and the plurality of sulfur compounds are two or more sulfur compounds selected from hydrogen disulfide, hydrogen trisulfide, cysteine ​​persulfide, and glutathione persulfide.

12. The method for measuring sulfur compounds according to claim 1, wherein the predetermined disease is COVID-19, and the plurality of sulfur compounds are two or more sulfur compounds selected from hydrogen sulfide, hydrogen disulfide, hydrogen trisulfide, cysteine ​​persulfide, and glutathione.

13. The method for measuring sulfur compounds according to claim 1, wherein the predetermined disease is depression and bipolar disorder, which are mental illnesses.

14. The method for measuring sulfur compounds according to claim 1, wherein the predetermined disease is depression, and the plurality of sulfur compounds are two or more sulfur compounds selected from hydrogen trisulfide, cysteine, and cysteine ​​persulfide.

15. A disease determination method comprising: a sulfur compound measurement step of analyzing samples obtained from the breath of multiple patients suffering from one of six diseases: breast cancer, gastrointestinal cancer, irritable bowel syndrome, COVID-19 infection, depression, and bipolar disorder, using a mass spectrometer to measure the amount of multiple types of sulfur compounds contained in the samples; an information acquisition step of obtaining information related to the six diseases by performing multivariate analysis on the amount of multiple types of sulfur compounds contained in the samples obtained from the breath of the multiple patients measured in the sulfur compound measurement step; and a determination step of determining whether or not the subject suffers from one of the six diseases, based on the results of analyzing a sample obtained from the breath of a subject using a mass spectrometer to measure the amount of multiple types of sulfur compounds contained in the sample and the information, wherein the multiple types of sulfur compounds include two or more sulfur compounds selected from 10 types of sulfur compounds: hydrogen sulfide, hydrogen disulfide, hydrogen trisulfide, hydrogen tetrasulfide, cysteine, cysteine ​​persulfide, glutathione, glutathione persulfide, sulfite, and thiosulfite.

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