Method for classifying bacteria or cells, method for classifying amino acid transport characteristics, method for assisting diagnosis of bacterial infection, and method for assisting diagnosis of cancer

Abstract

To provide a bacteria or cell classifying method, a classifying method for amino acid transportation properties, a bacteria infection diagnosis assisting method and a cancer diagnosis assisting method.SOLUTION: The inventors have discovered that under various conditions bacteria and cancer cells have specific amino acid accumulation properties (particularly amino acid transportation properties), thereby completing a bacteria or cell classifying method, a classifying method for amino acid transportation properties, a bacteria infection diagnosis assisting method and a cancer diagnosis assisting method.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for classifying bacteria or cells, a method for classifying the transport characteristics of amino acids, a method for assisting the diagnosis of bacterial infections, and a method for assisting the diagnosis of cancer.

Background Art

[0002] Bacterial infections caused by various bacteria often threaten the healthy life of humans. Bacterial infections are caused by the infection of hosts by pathogenic bacteria, opportunistic infectious bacteria, etc., and their pathological conditions are diverse depending on the type of causative bacteria (Non-Patent Document 1).

[0003] The onset of bacterial infections is related to two factors: the pathogenic factors of bacteria and the host's infection defense ability (Non-Patent Document 2). Important pathogenic factors related to the infectivity of bacteria include factors involved in the adhesion and invasion of host tissues, various enzymes that induce tissue damage, toxins that disrupt physiological and immune functions, and the like. On the other hand, as infection defense, there are innate immune responses centered on the phagocytosis of bacteria by neutrophils, macrophages, etc., and acquired immune responses that specifically eliminate bacteria antigen-specifically centered on T cells and B cells. Most bacteria are usually eliminated by these host immune responses and do not cause the onset of infectious diseases. However, when the host's immune response weakens, the lack of immune response to some pathogens, or the immune disruption effect by bacteria occurs, infectious diseases develop.

[0004] In severe acute infectious diseases such as invasive group A streptococcal infection, in addition to the host immune response, rapid bacterial growth observed at the site of infection and throughout the body also becomes a major problem. For example, necrotizing fasciitis is one of the symptoms of invasive group A streptococcal infection (Non-Patent Document 1), and this symptom is accompanied by rapid growth of group A streptococci and fascial necrosis at the site of infection, and elucidating the cause of this rapid growth of group A streptococci has become a major challenge. However, regarding the pathogenic factors of infectious bacteria, many studies have been conducted on the above-mentioned adhesion and invasion factors, various enzymes and toxins involved in tissue damage, etc., but there has been little progress in specific studies on the growth ability of bacteria at present.

[0005] The current method for detecting bacterial infections uses the bacterial culture method. However, since this method takes time until the test result is determined, it is not suitable for diagnosing severe infectious diseases with rapid symptom progression, high mortality, etc.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 10

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide novel means in a method for classifying bacteria or cells, a method for classifying amino acid transport characteristics, a method for assisting the diagnosis of bacterial infection, and a method for assisting the diagnosis of cancer.

Means for Solving the Problems

[0008] As a result of intensive studies, the present inventors have found that bacteria and cancer cells have specific amino acid accumulation characteristics (particularly amino acid transport characteristics) under various conditions, and have completed a method for classifying bacteria or cells, a method for classifying amino acid transport characteristics, a method for assisting the diagnosis of bacterial infection, and a method for assisting the diagnosis of cancer.

[0009] That is, the present invention is as follows. 1. A method for classifying bacteria or cells, including any one or more of the following steps, 1) Introducing a radioisotope-labeled amino acid into bacteria or cells in the presence or absence of Na + and measuring the uptake of the radioisotope-labeled amino acid into the cells in the presence or absence of Na+ dependency or Na + a step of identifying whether it is non-dependent, 2) a step of introducing a radioisotope-labeled amino acid into bacteria or cells in the presence of an inhibitor against an amino acid transporter to identify whether the uptake of the radioisotope-labeled amino acid into the cells is inhibited; 3) a step of introducing a radioisotope-labeled amino acid into bacteria or cells in the presence of an inhibitor against an amino acid transporter and in the presence or absence of Na + to identify whether the uptake of the radioisotope-labeled amino acid into the cells is inhibited; 4) a step of introducing a radioisotope-labeled amino acid into bacteria or cells in the presence of a system L-specific inhibitor to identify whether the uptake of the radioisotope-labeled amino acid into the cells is inhibited; 5) a step of introducing a radioisotope-labeled amino acid into bacteria or cells in the presence of a system L-specific inhibitor and in the presence or absence of Na + to identify whether the uptake of the radioisotope-labeled amino acid into the cells is inhibited; 6) a step of introducing a radioisotope-labeled amino acid into bacteria or cells in the presence of a system A-specific inhibitor to identify whether the uptake of the radioisotope-labeled amino acid into the cells is inhibited; 7) a step of introducing a radioisotope-labeled amino acid into bacteria or cells in the presence of a system A-specific inhibitor and in the presence or absence of Na + to identify whether the uptake of the radioisotope-labeled amino acid into the cells is inhibited; 8) a step of introducing a radioisotope-labeled amino acid into bacteria or cells in the presence of an unlabeled optically isomeric amino acid to identify whether the uptake of the radioisotope-labeled amino acid into the cells is inhibited, and 9) a step of introducing a radioisotope-labeled amino acid into bacteria or cells in the presence of an unlabeled optically isomeric amino acid and in the presence or absence of Na + to identify whether the uptake of the radioisotope-labeled amino acid into the cells is inhibited, A classification method comprising the above steps. 2.1) Administering Na in the presence or absence to a patient-derived sample + in the presence or absence of 3 H-L-Ala, 3 H-D-Ala, 3 H-L-Met or 3 H-D-Met, or administering a radioisotope-labeled natural L-amino acid or a radioisotope-labeled natural D-amino acid in the presence or absence of Na + 2) 3 When the accumulation rate of H-L-Ala, 3 H-D-Ala, 3 H-L-Met or 3 H-D-Met is lower in the presence of Na than in the absence of Na, and the difference in the accumulation rate is determined to be a Na-dependent transport property + compared to the presence of Na, + + A method for classifying the transport properties of amino acids of pathogenic bacteria, comprising 3.1) Administering to a patient-derived sample in the presence or absence of Na + in the presence or absence of 3 H-L-Ala, 3 H-D-Ala or a radioisotope-labeled natural amino acid and any one of L-Ala, D-Ala, L-Met or D-Met, or administering to a patient-derived sample in the presence or absence of Na + in the presence or absence of 3 H-L-Met, 3 H-D-Met or a radioisotope-labeled natural amino acid and any one of L-Met, D-Met, L-Ala or D-Ala, or administering to a patient-derived sample in the presence or absence of Na + in the presence or absence of a radio-labeled natural L-amino acid or a radio-labeled natural D-amino acid and its non-radioactive natural L-amino acid, non-radioactive natural D-amino acid, other non-radioactive natural L-amino acids or the non-radioactive natural D-amino acid of the corresponding optical isomer and 2) + The self-inhibition of L-Ala against the accumulation of H-L-Ala in the absence of Na 3 + ​​​​Non-dependent specific transport, in the presence or absence of Na + the difference in self-inhibition of L-Ala against the accumulation of H-L-Ala is Na + dependent specific transport, and in the absence of Na 3 the difference in self-inhibition of L-Ala and inhibition by D-Ala against the accumulation of H-L-Ala is Na + Non-dependent enantiomer-specific transport, and in the presence of Na + the difference in self-inhibition of L-Ala and inhibition by D-Ala against the accumulation of H-L-Ala minus the non-dependent enantiomer-specific transport is Na 3 judged to be dependent enantiomer-specific transport, + or, in the absence of Na + the self-inhibition of L-Met against the accumulation of H-L-Met is Na 3 Non-dependent specific transport, in the presence or absence of Na + the difference in self-inhibition of L-Met is Na + Dependent specific transport, and in the absence of Na the difference in self-inhibition of L-Met and inhibition by D-Met against the accumulation of H-L-Met is Na Non-dependent enantiomer-specific transport, and in the presence of Na + the difference in self-inhibition of L-Met and inhibition by D-Met against the accumulation of H-L-Met minus the non-dependent enantiomer-specific transport is Na 3 judged to be dependent enantiomer-specific transport, + or, in the absence of Na + the self-inhibition of non-radioactive natural-L-amino acid against the accumulation of radioisotope-labeled natural-L-amino acid is Na + Non-dependent specific transport, 3 in the presence or absence of Na + the difference in self-inhibition of L-Met is Na + Dependent specific transport, and in the absence of Na 3 the difference in self-inhibition of L-Met and inhibition by D-Met against the accumulation of H-L-Met is Na + Non-dependent enantiomer-specific transport, and in the presence of Na + the difference in self-inhibition of L-Met and inhibition by D-Met against the accumulation of H-L-Met minus the non-dependent enantiomer-specific transport is Na 3 judged to be dependent enantiomer-specific transport, + or, in the absence of Na + the self-inhibition of non-radioactive natural-L-amino acid against the accumulation of radioisotope-labeled natural-L-amino acid is Na Non-dependent specific transport, in the presence or absence of Na + the self-inhibition of non-radioactive natural-L-amino acid against the accumulation of radioisotope-labeled natural-L-amino acid is Na + Non-dependent specific transport,+ in the presence of or in the absence of Na + The difference in self-inhibition of non-radioactive natural-L-amino acids against the accumulation of radioisotope-labeled natural-L-amino acids in the presence or absence of Na + is Na-dependent specific transport, and Na + The difference between the self-inhibition of non-radioactive natural-L-amino acids and the inhibition by non-radioactive natural-D-amino acids against the accumulation of radioisotope-labeled natural-L-amino acids in the absence of Na + is Na-independent enantioselective transport, and Na + Among the difference between the self-inhibition of non-radioactive natural-L-amino acids and the inhibition by non-radioactive natural-D-amino acids against the accumulation of radioisotope-labeled natural-L-amino acids in the absence of Na + subtracting the Na-independent enantioselective transport is determined to be Na + dependent enantioselective transport, or, The Na + dependent specific transport, Na + independent specific transport, Na + dependent enantioselective transport, or Na + independent enantioselective transport ratio is determined to be a characteristic transport property, A method for classifying the amino acid transport characteristics of a pathogenic bacterium, comprising the above steps. 4.1) Administering H-L-Ala, + H-D-Ala or a radioisotope-labeled natural amino acid and either MeAIB or BCH to a patient-derived sample in the presence or absence of Na 3 or, administering H-L-Met, 3 H-D-Met or a radioisotope-labeled natural amino acid and either MeAIB or BCH to a patient-derived sample in the presence or absence of Na + or, administering H-L-Met, 3 H-D-Met or a radioisotope-labeled natural amino acid and either MeAIB or BCH to a patient-derived sample in the presence or absence of Na 3 or, administering H-L-Met, +administering a radiolabeled natural - L - amino acid or radiolabeled natural - D - amino acid in the presence or absence thereof, and its non - radiolabeled natural - L - amino acid, non - radiolabeled natural - D - amino acid, other non - radiolabeled natural - L - amino acids or the non - radiolabeled natural - D - amino acid of the corresponding optical isomer; and 2)Na + the inhibition of the accumulation of radiolabeled natural amino acid by MeAIB in the absence of Na is system PAT - specific transport, and Na + the inhibition of the accumulation of radiolabeled natural amino acid by MeAIB in the presence of Na is system A or system IMINO - specific transport, and Na + the inhibition of the accumulation of radiolabeled natural amino acid by BCH in the absence of Na is system L - specific transport, Na + the inhibition of the accumulation of radiolabeled natural amino acid by BCH in the presence of Na is system B 0,+ or system B 0 and is determined to be specific transport; or, Na + the self - inhibition of non - radiolabeled natural - L - amino acid on the accumulation of radiolabeled natural - L - amino acid in the absence of Na is Na + Na - independent specific transport, and Na + the difference in the self - inhibition of non - radiolabeled natural - L - amino acid on the accumulation of radiolabeled natural - L - amino acid in the presence or absence of Na is Na + Na - dependent specific transport, and Na + the difference between the self - inhibition of non - radiolabeled natural - L - amino acid and the inhibition by non - radiolabeled natural - D - amino acid on the accumulation of radiolabeled natural - L - amino acid in the absence of Na is Na + Na - independent optical isomer - specific transport, and Na + subtracting the Na - independent optical isomer - specific transport from the difference between the self - inhibition of non - radiolabeled natural - L - amino acid and the inhibition by non - radiolabeled natural - D - amino acid on the accumulation of radiolabeled natural - L - amino acid in the absence of Na gives Na + Among the difference between the self - inhibition of non - radiolabeled natural - L - amino acid and the inhibition by non - radiolabeled natural - D - amino acid on the accumulation of radiolabeled natural - L - amino acid in the absence of Na + the Na - independent optical isomer - specific transport subtracted is Na +A step determined to be dependent optical isomer-specific transport, or, Na-dependent specific transport for the accumulation of a specific radioisotope-labeled natural-L-amino acid, Na + -independent specific transport, Na + -independent optical isomer-specific transport, or a step determined that the ratio of Na + -dependent optical isomer-specific transport is characteristic of the transport property, + or a step determined that the ratio of Na -independent optical isomer-specific transport is characteristic of the transport property, including a method for classifying the transport properties of amino acids in cells. 5.1) A step determined that the ratio of Na + -dependent specific transport, Na + -independent specific transport, Na + -dependent optical isomer-specific transport, or Na + -independent optical isomer-specific transport for the accumulation of a specific radioisotope-labeled natural-L-amino acid in a patient-derived sample is characteristic of the transport property 2) A step determined that the ratio of Na + -dependent specific transport, Na + -independent specific transport, Na + -dependent optical isomer-specific transport, or Na + -independent optical isomer-specific transport for the accumulation of a specific radioisotope-labeled natural-D-amino acid in a patient-derived sample is characteristic of the transport property including a method for discriminating the transport properties of amino acids in bacteria or cells. 6. A method for assisting in the diagnosis of Escherichia coli infection, including the following steps, 1) A step of administering H-L-Ala to a patient-derived sample in the presence or absence of Na + ; 3 2) A step of administering H-L-Met to a patient-derived sample in the presence or absence of Na ; and + 3) The accumulation rate of H-L-Ala in the presence of Na 3 is higher than the accumulation rate of H-L-Ala in the absence of Na ; and + in the presence of Na 3 the accumulation rate of H-L-Ala, and + in the absence of Na 3 the accumulation rate of H-L-Ala, and + in the presence of Na3 The accumulation rate of H-L-Met in the presence or absence of Na + When it is comparable to the accumulation rate of H-L-Met in the absence of Na, a step of determining Escherichia coli infection, 3 A method for assisting in the diagnosis of Escherichia coli infection, comprising: A method for assisting in the diagnosis of Escherichia coli infection, comprising the following steps: 7. A method for assisting in the diagnosis of Escherichia coli infection, comprising the following steps: 1) Administering either H-L-Ala and L-Ala or H-L-Ala and D-Ala to a sample derived from a patient in the presence or absence of Na + in the presence or absence of Na 3 2) Administering either H-L-Met and L-Met or H-L-Met and D-Met to a sample derived from a patient in the presence or absence of Na 2) Administering either H-L-Met and L-Met or H-L-Met and D-Met to a sample derived from a patient in the presence or absence of Na + in the presence or absence of Na 3 3) When the inhibition of L-Ala is higher than that of D-Ala in the inhibition of the accumulation of H-L-Ala in the presence of Na, and the inhibition of L-Ala is comparable to that of D-Ala in the inhibition of the accumulation of H-L-Ala in the absence of Na, and the inhibition of L-Met is higher than that of D-Met in the inhibition of the accumulation of H-L-Met in the presence of Na, and the inhibition of L-Met is higher than that of D-Met in the inhibition of the accumulation of H-L-Met in the absence of Na, a step of determining Escherichia coli infection, 3) When the inhibition of L-Ala is higher than that of D-Ala in the inhibition of the accumulation of H-L-Ala in the presence of Na, and the inhibition of L-Ala is comparable to that of D-Ala in the inhibition of the accumulation of H-L-Ala in the absence of Na, and the inhibition of L-Met is higher than that of D-Met in the inhibition of the accumulation of H-L-Met in the presence of Na, and the inhibition of L-Met is higher than that of D-Met in the inhibition of the accumulation of H-L-Met in the absence of Na, a step of determining Escherichia coli infection, + in the presence of Na 3 When the inhibition of L-Ala is higher than that of D-Ala in the inhibition of the accumulation of H-L-Ala, and the inhibition of L-Ala is comparable to that of D-Ala in the inhibition of the accumulation of H-L-Ala in the absence of Na, and the inhibition of L-Met is higher than that of D-Met in the inhibition of the accumulation of H-L-Met in the presence of Na, and the inhibition of L-Met is higher than that of D-Met in the inhibition of the accumulation of H-L-Met in the absence of Na, a step of determining Escherichia coli infection, + in the absence of Na 3 When the inhibition of L-Ala is comparable to that of D-Ala in the inhibition of the accumulation of H-L-Ala, and the inhibition of L-Met is higher than that of D-Met in the inhibition of the accumulation of H-L-Met in the presence of Na, and the inhibition of L-Met is higher than that of D-Met in the inhibition of the accumulation of H-L-Met in the absence of Na, a step of determining Escherichia coli infection, + in the presence of Na 3 When the inhibition of L-Met is higher than that of D-Met in the inhibition of the accumulation of H-L-Met, and the inhibition of L-Met is higher than that of D-Met in the inhibition of the accumulation of H-L-Met in the absence of Na, a step of determining Escherichia coli infection, + in the absence of Na 3 When the inhibition of L-Met is higher than that of D-Met in the inhibition of the accumulation of H-L-Met, a step of determining Escherichia coli infection, A method for assisting in the diagnosis of Escherichia coli infection, comprising: 8. A method for assisting in the diagnosis of lung cancer, comprising the following steps: 1) Administering H-L-Ala to a sample derived from a patient in the presence or absence of Na + in the presence or absence of Na 3 2) Administering H-L-Met to a sample derived from a patient in the presence or absence of Na, and 2) Administering H-L-Met to a sample derived from a patient in the presence or absence of Na + in the presence or absence of Na 3 3) When the accumulation rate of H-L-Ala in the presence of Na is higher than that in the absence of Na, 3) When the accumulation rate of H-L-Ala in the presence of Na is higher than that in the absence of Na, + in the presence of Na 3 the accumulation rate of H-L-Ala + in the absence of Na 3Higher than the accumulation rate of H-L-Ala and in the presence of Na + in the presence of 3 the accumulation rate of H-L-Met is higher than that of H-L-Met in the absence of Na + in the absence of 3 a step of determining lung cancer when the accumulation rate of H-L-Met is higher than that of H-L-Met in the absence of Na, A method for assisting in the diagnosis of lung cancer, comprising: 9. A method for assisting in the diagnosis of lung cancer, comprising the following steps: 1) A step of administering either H-L-Ala or MeAIB or BCH to a sample derived from a patient in the presence or absence of Na + in the presence or absence of 3 ; 2) A step of administering either H-L-Met or MeAIB or BCH to a sample derived from a patient in the presence or absence of Na + in the presence or absence of 3 ; 3) When the inhibition of BCH is higher than the inhibition of MeAIB in the inhibition of the accumulation of H-L-Ala in the presence of Na, and the inhibition of BCH is observed but the inhibition of MeAIB is not observed in the inhibition of the accumulation of H-L-Ala in the absence of Na, and the inhibition of BCH is observed but the inhibition of MeAIB is not observed in the inhibition of the accumulation of H-L-Met in the presence of Na, and the inhibition of BCH is observed but the inhibition of MeAIB is not observed in the inhibition of the accumulation of H-L-Met in the absence of Na, a step of determining lung cancer + in the presence of 3 ; + in the absence of 3 ; + in the presence of 3 ; + in the absence of 3 ; a method for assisting in the diagnosis of lung cancer, comprising: 10. A method for assisting in the diagnosis of glioblastoma multiforme, comprising the following steps: 1) A step of administering H-L-Ala to a sample derived from a patient in the presence or absence of Na + in the presence or absence of 3 ; 2) A step of administering H-L-Met to a sample derived from a patient in the presence or absence of Na, and + in the presence or absence of 3 ; and 3) When the accumulation rate of H-L-Ala in the presence of Na is + in the presence of 3 higher than that of H-L-Ala in the absence of Na + ​In the absence of 3 higher than the accumulation rate of H-L-Ala and, in the presence of Na + In the presence of 3 when the accumulation rate of H-L-Met is similar to that of H-L-Met in the absence of Na + In the absence of 3 a step of determining glioblastoma multiforme when it is at the same level as the accumulation rate of H-L-Met, A method for assisting the diagnosis of glioblastoma multiforme, comprising 11. A method for assisting the diagnosis of glioblastoma multiforme, comprising the following steps: 1) A step of administering either H-L-Ala and MeAIB or BCH to a patient-derived sample in the presence or absence of Na + In the presence or absence of 3 H-L-Ala and either MeAIB or BCH; 2) A step of administering either H-L-Met and MeAIB or BCH to a patient-derived sample in the presence or absence of Na + In the presence or absence of 3 H-L-Met and either MeAIB or BCH; 3) When the BCH inhibition is higher than the MeAIB inhibition in the inhibition of the accumulation of H-L-Ala in the presence of Na + In the presence of 3 and the BCH inhibition is higher than the MeAIB inhibition in the inhibition of the accumulation of H-L-Ala in the absence of Na, and there is no MeAIB inhibition in the inhibition of the accumulation of H-L-Met in the presence of Na + In the absence of 3 and the BCH inhibition is higher than the MeAIB inhibition in the inhibition of the accumulation of H-L-Ala in the absence of Na, and there is no MeAIB inhibition in the inhibition of the accumulation of H-L-Met in the presence of Na + In the presence of 3 and there is no MeAIB inhibition in the inhibition of the accumulation of H-L-Met, and there is BCH inhibition but no MeAIB inhibition in the inhibition of the accumulation of H-L-Met in the absence of Na + In the absence of 3 a step of determining glioblastoma multiforme; A method for assisting the diagnosis of glioblastoma multiforme, comprising 12. A method for assisting the diagnosis of brain tumors, comprising the following steps: 1) A step of administering H-L-Ala to a patient-derived sample in the presence or absence of Na + In the presence or absence of 3 H-L-Ala; 2) A step of administering H-L-Met to a patient-derived sample in the presence or absence of Na + In the presence or absence of 3 H-L-Met, and 3) In the presence of Na + In the presence of 3The accumulation rate of H-L-Ala in the absence of Na + is higher than that of H-L-Ala in the presence of Na 3 and, when the accumulation rate of H-L-Met in the presence of Na + is higher than that of H-L-Met in the absence of Na 3 a step of determining that it is a brain tumor, + a method for assisting the diagnosis of a brain tumor, comprising the same. 3 13. A method for assisting the diagnosis of a brain tumor, comprising the following steps: 1) A step of administering either H-L-Ala and MeAIB or BCH to a patient-derived sample in the presence or absence of Na 2) A step of administering either H-L-Met and MeAIB or BCH to a patient-derived sample in the presence or absence of Na 3) A step of determining that it is a brain tumor when the BCH inhibition and the MeAIB inhibition are of the same degree in the inhibition of the accumulation of H-L-Ala in the presence of Na, and the BCH inhibition is observed but the MeAIB inhibition is not observed in the inhibition of the accumulation of H-L-Ala in the absence of Na, and the MeAIB inhibition is slight in the inhibition of the accumulation of H-L-Met in the presence of Na, and the BCH inhibition is observed but the MeAIB inhibition is not observed in the inhibition of the accumulation of H-L-Met in the absence of Na, + a method for assisting the diagnosis of a brain tumor, comprising the same. 3 14. 1) A step of administering either I-L-MIT, I-D-MIT or a radioisotope-labeled artificial amino acid and L-MIT, D-MIT and the natural amino acid L-Tyr or D-Tyr which is the labeled parent compound to a patient-derived sample in the presence of Na, or a step of administering either H-L-Tyr, 2) A step of administering either H-L-Met and MeAIB or BCH to a patient-derived sample in the presence or absence of Na + 3) A step of determining that it is a brain tumor when the BCH inhibition and the MeAIB inhibition are of the same degree in the inhibition of the accumulation of H-L-Ala in the presence of Na, and the BCH inhibition is observed but the MeAIB inhibition is not observed in the inhibition of the accumulation of H-L-Ala in the absence of Na, and the MeAIB inhibition is slight in the inhibition of the accumulation of H-L-Met in the presence of Na, and the BCH inhibition is observed but the MeAIB inhibition is not observed in the inhibition of the accumulation of H-L-Met in the absence of Na, 3 a method for assisting the diagnosis of a brain tumor, comprising the same. 3) A step of determining that it is a brain tumor when the BCH inhibition and the MeAIB inhibition are of the same degree in the inhibition of the accumulation of H-L-Ala in the presence of Na, and the BCH inhibition is observed but the MeAIB inhibition is not observed in the inhibition of the accumulation of H-L-Ala in the absence of Na, and the MeAIB inhibition is slight in the inhibition of the accumulation of H-L-Met in the presence of Na, and the BCH inhibition is observed but the MeAIB inhibition is not observed in the inhibition of the accumulation of H-L-Met in the absence of Na, + a method for assisting the diagnosis of a brain tumor, comprising the same. 3 3) When the BCH inhibition and the MeAIB inhibition are of the same degree in the inhibition of the accumulation of H-L-Ala in the presence of Na, and the BCH inhibition is observed but the MeAIB inhibition is not observed in the inhibition of the accumulation of H-L-Ala in the absence of Na, and the MeAIB inhibition is slight in the inhibition of the accumulation of H-L-Met in the presence of Na, and the BCH inhibition is observed but the MeAIB inhibition is not observed in the inhibition of the accumulation of H-L-Met in the absence of Na, + a step of determining that it is a brain tumor, 3 a method for assisting the diagnosis of a brain tumor, comprising the same. + 14. 1) A step of administering either I-L-MIT, I-D-MIT or a radioisotope-labeled artificial amino acid and L-MIT, D-MIT and the natural amino acid L-Tyr or D-Tyr which is the labeled parent compound to a patient-derived sample in the presence of Na, or a step of administering either H-L-Tyr, 3 2) A step of administering either H-L-Met and MeAIB or BCH to a patient-derived sample in the presence or absence of Na + 3) A step of determining that it is a brain tumor when the BCH inhibition and the MeAIB inhibition are of the same degree in the inhibition of the accumulation of H-L-Ala in the presence of Na, and the BCH inhibition is observed but the MeAIB inhibition is not observed in the inhibition of the accumulation of H-L-Ala in the absence of Na, and the MeAIB inhibition is slight in the inhibition of the accumulation of H-L-Met in the presence of Na, and the BCH inhibition is observed but the MeAIB inhibition is not observed in the inhibition of the accumulation of H-L-Met in the absence of Na, 3 a method for assisting the diagnosis of a brain tumor, comprising the same. 14. 1) A step of administering either I-L-MIT, I-D-MIT or a radioisotope-labeled artificial amino acid and L-MIT, D-MIT and the natural amino acid L-Tyr or D-Tyr which is the labeled parent compound to a patient-derived sample in the presence of Na, or a step of administering either H-L-Tyr, 14. 1) A step of administering either I-L-MIT, I-D-MIT or a radioisotope-labeled artificial amino acid and L-MIT, D-MIT and the natural amino acid L-Tyr or D-Tyr which is the labeled parent compound to a patient-derived sample in the presence of Na, or a step of administering either H-L-Tyr, + 2) A step of administering either H-L-Met and MeAIB or BCH to a patient-derived sample in the presence or absence of Na 125 3) A step of determining that it is a brain tumor when the BCH inhibition and the MeAIB inhibition are of the same degree in the inhibition of the accumulation of H-L-Ala in the presence of Na, and the BCH inhibition is observed but the MeAIB inhibition is not observed in the inhibition of the accumulation of H-L-Ala in the absence of Na, and the MeAIB inhibition is slight in the inhibition of the accumulation of H-L-Met in the presence of Na, and the BCH inhibition is observed but the MeAIB inhibition is not observed in the inhibition of the accumulation of H-L-Met in the absence of Na, 125 a method for assisting the diagnosis of a brain tumor, comprising the same. + 14. 1) A step of administering either I-L-MIT, I-D-MIT or a radioisotope-labeled artificial amino acid and L-MIT, D-MIT and the natural amino acid L-Tyr or D-Tyr which is the labeled parent compound to a patient-derived sample in the presence of Na, or a step of administering either H-L-Tyr, 3 2) A step of administering either H-L-Met and MeAIB or BCH to a patient-derived sample in the presence or absence of Na 3Administering either H-D-Tyr or a radioisotope-labeled natural amino acid and either L-Tyr, D-Tyr, or the unlabeled form of the labeled artificial amino acid, L-MIT or D-MIT, or, in a patient-derived sample, Na + administering a radioisotope-labeled natural -L-amino acid or radioisotope-labeled natural -D-amino acid and its non-radioactive natural -L-amino acid, non-radioactive natural -D-amino acid, other non-radioactive natural -L-amino acids, or the non-radioactive natural -D-amino acid of the corresponding optical isomer, in the presence of 2) The self-inhibition of L-MIT on the accumulation of + I-L-MIT in the presence of 125 Na is the specific transport of this artificial amino acid, and the difference between the self-inhibition of L-MIT on the accumulation of + I-L-MIT and the inhibition by D-MIT in the presence of 125 Na is the specific transport of optical isomers, and the self-inhibition of D-MIT on the accumulation of + I-D-MIT in the presence of 125 Na is the specific transport of this artificial amino acid, and the difference between the self-inhibition of D-MIT on the accumulation of + I-D-MIT and the inhibition by L-MIT in the presence of 125 Na is determined to be the specific transport of optical isomers. Or, The self-inhibition of L-Tyr on the accumulation of + H-L-Tyr in the presence of 3 Na is the specific transport of this natural amino acid, and the difference between the self-inhibition of L-Tyr on the accumulation of + H-L-Tyr and the inhibition by D-Tyr in the presence of 3 Na is the specific transport of optical isomers, and the difference between the self-inhibition of L-Tyr on the accumulation of + H-L-Tyr and the inhibition by L-MIT in the presence of 3 Na is determined to be the specific transport of natural amino acid-selective optical isomers. Or, The difference between the inhibition by D-Tyr and the inhibition by D-MIT on the accumulation of + H-L-Tyr in the presence of 3 Na is determined to be the non-selective transport of natural amino acid non-specific optical isomers. or a step of determining that the transport characteristics are characterized by the magnitude of the specific transport of the natural amino acid, optical isomer-specific transport, selective optical isomer-specific transport of the natural amino acid, or the ratio of selective optical isomer-specific transport of the natural amino acid to the accumulation of a specific radioisotope-labeled natural-L-amino acid A method for classifying the amino acid transport characteristics of bacteria, comprising: 15. A method for assisting in the diagnosis of pathogenic bacterial infection in Escherichia coli, comprising the following steps: 1) a step of administering Na + in the presence or absence of 3 H-L-Met to a sample derived from a patient, and 2) a step of determining that the patient is infected with pathogenic Escherichia coli when the accumulation rate of L-Met in the presence of Na + is higher than the accumulation rate of + H-L-Met in the absence of Na 3 ; or 1) a step of administering Na + in the presence or absence of 3 H-L-Ala and MeAIB or BCH to a sample derived from a patient, and 2) a step of determining that the patient is infected with pathogenic Escherichia coli when the inhibition of the accumulation of + H-L-Ala in the presence of Na 3 is lower than the inhibition of the accumulation of + H-L-Ala in the absence of Na 3 ; or 1) a step of administering Na + in the absence of 3 H-L-Ala and D-Ala to a sample derived from a patient, and 2) a step of determining that the patient is infected with pathogenic Escherichia coli when the accumulation rate of L-Ala in the absence of Na + is 20% or less compared to the control A method for assisting in the diagnosis of pathogenic bacterial infection in Escherichia coli, comprising: [Effect of the Invention]

[0010] The present invention can provide a novel method for classifying bacteria or cells, a method for classifying the transport characteristics of amino acids, a method for assisting the diagnosis of bacterial infections, and a method for assisting the diagnosis of cancer.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Mode for Carrying Out the Invention

[0012] (Object of the Present Invention) The present invention relates to a method for classifying bacteria or cells, a method for classifying the transport characteristics of amino acids, a method for assisting in the diagnosis of bacterial infections, and a method for assisting in the diagnosis of cancer.

[0013] (Method for classifying bacteria or cells) The method for classifying bacteria or cells of the present invention (hereinafter sometimes abbreviated as "the classification method of the present invention") identifies the type of bacteria or the type of cells by combining any one or more of the following steps. 1) Introduce a radioisotope-labeled amino acid into bacteria or cells in the presence or absence of Na + to identify whether the uptake of the radioisotope-labeled amino acid into the cells is Na + dependent or Na + independent. Further, the accumulation rate of amino acids under Na + dependence may be compared with the accumulation rate of amino acids under Na + independence. 2) Introduce a radioisotope-labeled amino acid into bacteria or cells in the presence of an inhibitor against an amino acid transporter to identify whether the uptake of the radioisotope-labeled amino acid into the cells is inhibited. The amino acid transporter is not particularly limited, but both Na + dependent and Na + independent amino acid transporters are targeted. Further, system A (ATA1, ATA2, ATA3, etc.), system L (LAT1, LAT2, LAT3, LAT4, etc.) can be exemplified. 3) Introduce a radioisotope-labeled amino acid into bacteria or cells in the presence of an inhibitor against an amino acid transporter and in the presence or absence of Na + to identify whether the uptake of the radioisotope-labeled amino acid into the cells is inhibited. The inhibitor against the amino acid transporter is not particularly limited, but known BCH (2-Aminobicyclo[2.2.1]heptane-2-carboxylic acid), MeAIB (α-methylaminoisobutyric acid), etc. can be exemplified. 4) Introducing a radioisotope-labeled amino acid into bacteria or cells in the presence of a system L-specific inhibitor to identify whether the intracellular uptake of the radioisotope-labeled amino acid is inhibited. 5) Introducing a radioisotope-labeled amino acid into bacteria or cells in the presence of a system L-specific inhibitor and in the presence or absence of Na + to identify whether the intracellular uptake of the radioisotope-labeled amino acid is inhibited. 6) Introducing a radioisotope-labeled amino acid into bacteria or cells in the presence of a system A-specific inhibitor to identify whether the intracellular uptake of the radioisotope-labeled amino acid is inhibited. 7) Introducing a radioisotope-labeled amino acid into bacteria or cells in the presence of a system A-specific inhibitor and in the presence or absence of Na + to identify whether the intracellular uptake of the radioisotope-labeled amino acid is inhibited. 8) Introducing a radioisotope-labeled amino acid into bacteria or cells in the presence of an unlabeled optically isomeric amino acid to identify whether the intracellular uptake of the radioisotope-labeled amino acid is inhibited. 9) Introducing a radioisotope-labeled amino acid into bacteria or cells in the presence of an unlabeled optically isomeric amino acid and in the presence or absence of Na + to identify whether the intracellular uptake of the radioisotope-labeled amino acid is inhibited.

[0014] (Method for classifying amino acid transport characteristics) The method for classifying the transport characteristics of amino acids of the present invention classifies the transport characteristics of amino acids in humans, bacteria, cells, etc. (particularly, pathogenic bacteria, cancer cells) by combining any one or more of the following steps, methods, etc. In particular, in the method for classifying the transport characteristics of amino acids of the present invention, at least, Na + -dependent transport characteristics, Na + -independent specific transport, Na + -independent optically isomer-specific transport, Na +Dependent optical isomer-specific transport, system PAT-specific transport, system A-specific transport, system IMINO-specific transport, system L-specific transport, system B 0,+ Specific transport, system B 0 Can be classified as specific transport.

[0015] 〇Example 1 of classification method for transport characteristics of amino acids 1) Na is added to the patient-derived sample + In the presence or absence of 3 H-L-Ala, 3 H-D-Ala, 3 H-L-Met or 3 H-D-Met is administered, or a radioactive isotope-labeled natural-L-amino acid or a radioactive isotope-labeled natural-D-amino acid is administered in the presence or absence of Na + 2) 3 H-L-Ala, 3 H-D-Ala, 3 H-L-Met or 3 H-D-Met accumulation rate, when the accumulation rate in the absence of Na is lower compared to in the presence of Na + Na + The difference in the accumulation rate is determined to be Na + Dependent transport characteristics.

[0016] 〇Example 2 of classification method for transport characteristics of amino acids 1) Na is added to the patient-derived sample + In the presence or absence of 3 H-L-Ala, 3 H-D-Ala or a radioactive isotope-labeled natural amino acid and either L-Ala, D-Ala, L-Met or D-Met are administered, or Na is added to the patient-derived sample + In the presence or absence of 3 H-L-Met, 3 H-D-Met or a radioactive isotope-labeled natural amino acid and either L-Met, D-Met, L-Ala or D-Ala are administered, or Na is added to the patient-derived sample + ​administering a radiolabeled natural - L - amino acid or radiolabeled natural - D - amino acid, and its non - radiolabeled natural - L - amino acid, non - radiolabeled natural - D - amino acid, other non - radiolabeled natural - L - amino acids or the non - radiolabeled natural - D - amino acid of the corresponding optical isomer, in the presence or absence thereof; 2)Na + in the absence of 3 The self - inhibition of L - Ala on the accumulation of H - L - Ala is Na + independent specific transport, and Na + in the presence or Na + in the absence of 3 The difference in the self - inhibition of L - Ala on the accumulation of H - L - Ala is Na + dependent specific transport, and, Na + in the absence of 3 The difference between the self - inhibition of L - Ala on the accumulation of H - L - Ala and the inhibition of D - Ala is Na + independent optical isomer - specific transport, and Na + in the presence of 3 Among the difference between the self - inhibition of L - Ala on the accumulation of H - L - Ala and the inhibition of D - Ala, the part that subtracts the Na + independent optical isomer - specific transport is determined to be Na + dependent optical isomer - specific transport; or, Na + in the absence of 3 The self - inhibition of L - Met on the accumulation of H - L - Met is Na + independent specific transport, and Na + in the presence or Na + in the absence of 3 The difference in the self - inhibition of L - Met on the accumulation of H - L - Met is Na + dependent specific transport, and, Na + in the absence of 3 The difference between the self - inhibition of L - Met on the accumulation of H - L - Met and the inhibition of D - Met is Na + independent optical isomer - specific transport, and Na + in the presence of 3The difference between the self-inhibition of L-Met and the inhibition of D-Met on the accumulation of H-L-Met, minus the Na + independent optical isomer-specific transport, is determined to be the Na + dependent optical isomer-specific transport process; or Na + The self-inhibition of non-radioactive natural-L-amino acid on the accumulation of radioisotope-labeled natural-L-amino acid in the absence of Na + is independent specific transport, and Na + the difference between the self-inhibition of non-radioactive natural-L-amino acid on the accumulation of radioisotope-labeled natural-L-amino acid in the presence or + absence of Na + is dependent specific transport, and + the difference between the self-inhibition of non-radioactive natural-L-amino acid and the inhibition of non-radioactive natural-D-amino acid on the accumulation of radioisotope-labeled natural-L-amino acid in the absence of Na + is independent optical isomer-specific transport, and Na + the difference between the self-inhibition of non-radioactive natural-L-amino acid and the inhibition of non-radioactive natural-D-amino acid on the accumulation of radioisotope-labeled natural-L-amino acid in the absence of Na + minus the Na + independent optical isomer-specific transport is determined to be the Na dependent optical isomer-specific transport process; or the Na + dependent specific transport, Na + independent specific transport, Na + dependent optical isomer-specific transport, or + the proportion of independent optical isomer-specific transport being characteristic transport properties is determined as a process.

[0017] 〇Example 3 of the classification method for the transport characteristics of amino acids 1) To a patient-derived sample, Na + in the presence or absence 3 H-L-Ala, 3The step of administering either H-D-Ala or a radioisotope-labeled natural amino acid and either MeAIB or BCH, or Na in a patient-derived sample + in the presence or absence of 3 H-L-Met, 3 the step of administering either H-D-Met or a radioisotope-labeled natural amino acid and either MeAIB or BCH, or Na in a patient-derived sample + The step of administering a radioactively labeled natural-L-amino acid or a radioactively labeled natural-D-amino acid and its non-radioactive natural-L-amino acid, non-radioactive natural-D-amino acid, other non-radioactive natural-L-amino acids or the non-radioactive natural-D-amino acid of the corresponding optical isomer in the presence or absence of and 2) Na + The inhibition of MeAIB on the accumulation of radioisotope-labeled natural amino acids in the absence of Na is system PAT-specific transport, and Na + The inhibition of MeAIB on the accumulation of radioisotope-labeled natural amino acids in the presence of Na is system A or system IMINO-specific transport, and + The inhibition of BCH on the accumulation of radioisotope-labeled natural amino acids in the absence of Na is system L-specific transport, and Na + The inhibition of BCH on the accumulation of radioisotope-labeled natural amino acids in the presence of Na is system B 0,+ or system B 0 is determined to be specific transport or Na + The self-inhibition of non-radioactive natural-L-amino acids on the accumulation of radioisotope-labeled natural-L-amino acids in the absence of Na is Na + non-dependent specific transport, and Na + the difference in the self-inhibition of non-radioactive natural-L-amino acids on the accumulation of radioisotope-labeled natural-L-amino acids in the presence or absence of Na is Na + dependent specific transport, and + Na +The difference between the self-inhibition of non-radioactive natural-L-amino acids and the inhibition of non-radioactive natural-D-amino acids on the accumulation of radioisotope-labeled natural-L-amino acids in the absence of Na + is Na + independent optical isomer-specific transport, and the Na + independent optical isomer-specific transport is subtracted from the difference between the self-inhibition of non-radioactive natural-L-amino acids and the inhibition of non-radioactive natural-D-amino acids on the accumulation of radioisotope-labeled natural-L-amino acids in the absence of Na + is determined to be a step of Na or the Na + dependent specific transport, Na + independent specific transport, Na + dependent optical isomer-specific transport, or Na + the ratio of independent optical isomer-specific transport is determined to be a characteristic transport property, A method for classifying the transport characteristics of amino acids in cells, including

[0018] 〇Example 4 of the method for classifying the transport characteristics of amino acids 1) Administer I-L-MIT, + in the presence of Na 125 I-D-MIT or a radioisotope-labeled artificial amino acid and either the natural amino acid L-Tyr or D-Tyr which is the labeled parent compound, or administer to the patient-derived sample either 125 H-L-Tyr, + H-D-Tyr or a radioisotope-labeled natural amino acid and either the unlabeled L-MIT or D-MIT which is the unlabeled form of the labeled artificial amino acid, or administer to the patient-derived sample either 3 H-L-Tyr, 3 H-D-Tyr or a radioisotope-labeled natural amino acid and either the unlabeled L-MIT or D-MIT which is the unlabeled form of the labeled artificial amino acid, or administer to the patient-derived sample a radioactive-labeled natural-L-amino acid or radioactive-labeled natural-D-amino acid and its non-radioactive natural-L-amino acid, non-radioactive natural-D-amino acid, other non-radioactive natural-L-amino acids or non-radioactive natural-D-amino acids corresponding to the optical isomers in the presence of Na + ​ 2) Na + in the presence of 125 self-inhibition of L-MIT against the accumulation of I-L-MIT is the specific transport of the artificial amino acid, and Na + in the presence of 125 the difference between self-inhibition of L-MIT against the accumulation of I-L-MIT and inhibition by D-MIT is the enantioselective transport, and Na + in the presence of 125 self-inhibition of D-MIT against the accumulation of I-D-MIT is the specific transport of the artificial amino acid, and Na + in the presence of 125 a step of determining that the difference between self-inhibition of D-MIT against the accumulation of I-D-MIT and inhibition by L-MIT is the enantioselective transport, or, Na + in the presence of 3 self-inhibition of L-Tyr against the accumulation of H-L-Tyr is the specific transport of the natural amino acid, and Na + in the presence of 3 the difference between self-inhibition of L-Tyr against the accumulation of H-L-Tyr and inhibition by D-Tyr is the enantioselective transport, and Na + in the presence of 3 a step of determining that the difference between self-inhibition of L-Tyr against the accumulation of H-L-Tyr and inhibition by L-MIT is the natural amino acid-selective enantioselective transport, or, Na + in the presence of 3 a step of determining that the difference between inhibition by D-Tyr and inhibition by D-MIT against the accumulation of H-L-Tyr is the natural amino acid-nonselective enantioselective non-specific transport, or, a step of determining that the specific transport of the natural amino acid, enantioselective transport, the natural amino acid-selective enantioselective transport, or the ratio of the natural amino acid-selective enantioselective transport with respect to the accumulation of a specific radioisotope-labeled natural-L-amino acid is the characteristic transport property.

[0019] (Method for discriminating amino acid transport properties) The method for discriminating the transport characteristics of amino acids of the present invention can discriminate the transport characteristics of amino acids in humans, bacteria, cells, etc. (particularly, pathogenic bacteria, cancer cells) by combining any one or more of the following steps, methods, etc. 1) The Na + -dependent specific transport, Na + -independent specific transport, Na + -dependent enantiomer-specific transport, or the proportion of Na + -independent enantiomer-specific transport being large or small is determined as a characteristic transport property in the step of determining. 2) The Na + -dependent specific transport, Na + -independent specific transport, Na + -dependent enantiomer-specific transport, or the proportion of Na + -independent enantiomer-specific transport being large or small is determined as a characteristic transport property in the step of determining.

[0020] (Application of the method for classifying / discriminating the transport characteristics of amino acids) As an application of the method for classifying / discriminating the transport characteristics of amino acids of the present invention, by classifying the transport characteristics of amino acids in bacteria, cells, etc., it can be used for the following applications. 1) By classifying the transport characteristics of amino acids in bacteria, cells, etc., it can be determined which therapeutic agents are effective. 2) By comparing the transport characteristics of amino acids in unknown bacteria, cells, etc. with those of known bacteria, cells, etc., the unknown bacteria, cells, etc. can be identified (see the following method for assisting in the diagnosis of bacterial infections and method for assisting in the diagnosis of cancer).

[0021] (Method for assisting in the diagnosis of bacterial infections) The method for assisting in the diagnosis of bacterial infections of the present invention includes the following steps according to the results of this example. 〇Example 1 of the method for assisting in the diagnosis of bacterial infections of the present invention 1) Na + is present or absent in the patient-derived sample 3The step of administering H-L-Ala. 2) Na in a patient-derived sample + In the presence or absence of 3 The step of administering H-L-Met. 3) Na + In the presence of 3 The accumulation rate of H-L-Ala is higher than that of H-L-Ala in the absence of Na + In the absence of 3 And the accumulation rate of H-L-Met in the presence of Na is comparable to that of H-L-Met in the absence of Na + In the presence of 3 The accumulation rate of H-L-Met + In the absence of 3 The step of determining Escherichia coli infection when the accumulation rate of H-L-Met is comparable to that of H-L-Met in the absence of Na. 〇 Example 2 of the method for assisting in the diagnosis of bacterial infection of the present invention 1) Either H-L-Ala or L-Ala or D-Ala is administered to a patient-derived sample in the presence or absence of Na + In the presence or absence of 3 The step of administering. 2) Either H-L-Met or L-Met or D-Met is administered to a patient-derived sample in the presence or absence of Na + In the presence or absence of 3 The step of administering. 3) In the inhibition of the accumulation of H-L-Ala in the presence of Na, when the inhibition by L-Ala is higher than the inhibition by D-Ala, and in the absence of Na + In the presence of 3 When the inhibition by L-Ala and the inhibition by D-Ala are comparable in the inhibition of the accumulation of H-L-Ala, and in the presence of Na + In the absence of 3 When the inhibition by L-Met is higher than the inhibition by D-Met in the inhibition of the accumulation of H-L-Met, and in the absence of Na + In the presence of 3 When the inhibition by L-Met is higher than the inhibition by D-Met in the inhibition of the accumulation of H-L-Met + In the absence of 3 The step of determining Escherichia coli infection when the inhibition by L-Met is higher than the inhibition by D-Met in the inhibition of the accumulation of H-L-Met.

[0022] (Method for assisting in the diagnosis of bacterial infection) The method for assisting in the diagnosis of pathogenic bacterial infection in Escherichia coli of the present invention includes the following steps according to the results of this example. 〇Example 1 of the method for assisting in the diagnosis of pathogenic bacterial infection in Escherichia coli of the present invention 1) Administer H-L-Met to the sample derived from the patient in the presence or absence of Na + 3 . 2) When the accumulation rate of L-Met in the presence of Na + is higher than the accumulation rate of H-L-Met in the absence of Na + , it is determined that there is an infection with the pathogenic bacterium Escherichia coli 3 . 〇Example 2 of the method for assisting in the diagnosis of pathogenic bacterial infection in Escherichia coli of the present invention 1) Administer H-L-Ala and MeAIB or BCH to the sample derived from the patient in the presence or absence of Na + 3 . 2) When the inhibition of the accumulation of H-L-Ala in the presence of Na + is lower than the inhibition of the accumulation of H-L-Ala in the absence of Na 3 , it is determined that there is an infection with the pathogenic bacterium Escherichia coli + 3 . 〇Example 3 of the method for assisting in the diagnosis of pathogenic bacterial infection in Escherichia coli of the present invention 1) Administer H-L-Ala and D-Ala to the sample derived from the patient in the absence of Na + 3 . 2) When the accumulation rate of L-Ala in the absence is 20% or less (or 15% or less, 10% or less, 5% or less) compared to the control, it is determined that there is an infection with the pathogenic bacterium Escherichia coli + .

[0023] (Method for assisting in the diagnosis of cancer) The method for assisting in the diagnosis of cancer of the present invention includes the following steps according to the results of this example

[0024] (Method for assisting in the diagnosis of lung cancer) 〇Example 1 of the method for assisting in the diagnosis of lung cancer 1) Administer H-L-Ala to the sample derived from the patient in the presence or absence of Na + 3 . 2) Administer Na to the sample derived from the patient + ​​​​​in the presence or absence 3 a step of administering H-L-Met. 3) Na + in the presence of 3 the accumulation rate of H-L-Ala is higher than that of H-L-Ala in the absence of Na + in the absence of 3 and the accumulation rate of H-L-Met in the presence of Na is higher than that of H-L-Met in the absence of Na, a step of determining that it is lung cancer. + in the presence of 3 the accumulation rate of H-L-Met + in the absence of 3 a step of determining that it is lung cancer when the accumulation rate of H-L-Met is high as compared with the accumulation rate of H-L-Met in the absence of Na. 〇Example 2 of a method for assisting in the diagnosis of lung cancer 1) Na is administered to a sample derived from a patient + in the presence or absence 3 with either H-L-Ala and MeAIB or BCH. 2) Na is administered to a sample derived from a patient + in the presence or absence 3 with either H-L-Met and MeAIB or BCH. 3) Na + in the presence of 3 when the inhibition of BCH is higher than the inhibition of MeAIB in the inhibition of the accumulation of H-L-Ala, and + in the absence of 3 the inhibition of BCH is observed but the inhibition of MeAIB is not observed in the inhibition of the accumulation of H-L-Ala, and + in the presence of 3 the inhibition of BCH is observed but the inhibition of MeAIB is not observed in the inhibition of the accumulation of H-L-Met, and + in the absence of 3 a step of determining that it is glioblastoma multiforme when the inhibition of BCH is observed but the inhibition of MeAIB is not observed in the inhibition of the accumulation of H-L-Met.

[0025] (Method for assisting in the diagnosis of glioblastoma multiforme) 〇Example 1 of a method for assisting in the diagnosis of glioblastoma multiforme 1) Na is administered to a sample derived from a patient + in the presence or absence 3 with H-L-Ala. 2) Na is administered to a sample derived from a patient +in the presence or absence 3 The step of administering H-L-Met. 3) Na + in the presence of 3 The accumulation rate of H-L-Ala is higher than that of H-L-Ala in the absence of Na + in the absence of 3 and, the accumulation rate of H-L-Met in the presence of Na is comparable to that of H-L-Met in the absence of Na, and + in the presence of 3 the accumulation rate of H-L-Met is + in the absence of 3 When the accumulation rate of H-L-Met is comparable to that of H-L-Met in the absence of Na, it is determined as glioblastoma multiforme. 〇Example 2 of the method for assisting the diagnosis of glioblastoma multiforme 1) Administer either H-L-Ala and MeAIB or BCH to the patient-derived sample in the presence or absence of Na + in the presence or absence 3 The step of administering either H-L-Ala and MeAIB or BCH. 2) Administer either H-L-Met and MeAIB or BCH to the patient-derived sample in the presence or absence of Na + in the presence or absence 3 The step of administering either H-L-Met and MeAIB or BCH. 3) Na + in the presence of 3 In the inhibition of the accumulation of H-L-Ala, the inhibition by BCH is higher than the inhibition by MeAIB, and + in the absence of 3 in the inhibition of the accumulation of H-L-Ala, the inhibition by BCH is higher than the inhibition by MeAIB, and + in the presence of 3 in the inhibition of the accumulation of H-L-Met, there is no inhibition by MeAIB, and + in the absence of 3 When in the inhibition of the accumulation of H-L-Met, the inhibition by BCH is observed but there is no inhibition by MeAIB, it is determined as glioblastoma multiforme The step of being determined.

[0026] (Method for assisting the diagnosis of brain tumor) 〇Example 1 of the method for assisting the diagnosis of brain tumor 1) Administer H-L-Ala to the patient-derived sample in the presence or absence of Na + in the presence or absence 3 The step of administering H-L-Ala. 2) Administer Na to the patient-derived sample +in the presence or absence 3 administering H-L-Met 3) Na + in the presence of 3 the accumulation rate of H-L-Ala is higher than that of H-L-Ala in the absence of Na and + in the absence of 3 and the accumulation rate of H-L-Met in the presence of Na is higher than that of H-L-Met in the absence of Na, then determining that it is a brain tumor + in the presence of 3 the accumulation rate of H-L-Met + in the absence of 3 and the accumulation rate of H-L-Met is higher than that of H-L-Met in the absence of Na, then determining that it is a brain tumor 〇Example 2 of the method for assisting in the diagnosis of brain tumors 1) Administer either H-L-Ala and MeAIB or BCH to the patient-derived sample in the presence or absence of Na + in the presence or absence 3 of Na 2) Administer either H-L-Met and MeAIB or BCH to the patient-derived sample in the presence or absence of Na + in the presence or absence 3 of Na 3) Na + in the presence of 3 if the inhibition of H-L-Ala accumulation by BCH and MeAIB is about the same and + in the absence of 3 Na, BCH inhibition of H-L-Ala accumulation is observed but MeAIB inhibition is not, and + in the presence of 3 Na, MeAIB inhibition of H-L-Met accumulation is slight and + in the absence of 3 Na, BCH inhibition of H-L-Met accumulation is observed but MeAIB inhibition is not, then determining that it is a brain tumor

[0027] (radioisotope) The radioisotope-labeled amino acid used in the present invention is one in which a radioisotope is bound to an amino acid. The radioisotope is not particularly limited, but examples include 3-hydrogen ( 3 H), 11-carbon ( 11 C), 15-oxygen ( 15 O), 18-fluorine (18 F), 32-Phosphorus( 32 P), 59-Iron( 59 Fe), 67-Copper( 67 Cu), 67-Gallium( 67 Ga), 81m-Krypton( 81m Kr), 81-Rubidium( 81 Rb), 89-Strontium( 89 Sr), 90-Yttrium( 90 Y), 99m-Technetium( 99m Tc), 111-Indium( 111 In), 123-Iodine( 123 I), 125-Iodine( 125 I), 131-Iodine( 131 I), 133-Xenon( 133 Xe), 117m-Samarium( 117m Sm), 153-Samarium( 153 Sm), 186-Rhenium( 186 Re), 188-Rhenium( 188 Re), 201-Thallium( 201 Tl), 212-Bismuth( 212 Bi), 213-Bismuth( 212 Bi) and 211-Astatine( 211 At) etc. can be mentioned.

[0028] (Amino acid) The amino acid labeled with the radioisotope used in the present invention is not particularly limited, and examples include neutral amino acids such as D-alanine, D-methionine, Glycine, L-alanine, L-tyrosine and L-methionine, acidic natural amino acid L-glutamic acid, basic natural amino acids such as L-lysine and L-histidine.

[0029] (Radioisotope-labeled amino acid) The radioisotope-labeled amino acid used in the present invention may be any that can be detected after being administered to cells, bacterial cells, the body, etc. As a specific example, 3 H, 18 F,125 Amino acids such as D-alanine, D-methionine, glycine, L-tyrosine, L-methionine, L-glutamic acid, L-lysine, and L-histidine, which are acidic natural amino acids, bound to radioactive isotopes such as I, salts thereof, and derivatives thereof, etc. are mentioned. For example, [2,3- 3 H]-D-alanine, [metyl- 3 H]-D-methionine, [2- 3 H]-glycine ( 3 H-Gly), [2,3- 3 H]-L-alanine ( 3 H-L-Ala), [ring3,5- 3 H]-L-tyrosine ( 3 H-L-Tyr), [methyl- 3 H]-L-methionine ( 3 H-L-Met), [2,3,4- 3 H]-L-glutamic acid ( 3 H-L-Glu), [4,5- 3 H]-L-lysine ( 3 H-L-Lys), [ring2,5- 3 H]-L-histidine ( 3 H-L-His), [3- 3 H-L-His) labeled with radioactive iodine 125 I, [3- 125 I]-L-tyrosine ( 125 I-L-Tyr), [5- 125 I]-L-histidine ( 125 I-L-His), [S-methyl- 11 C]-L-methionine, etc. labeled with radioactive carbon (carbon 11: 11 C) are mentioned.

[0030] (Classification results of the amino acid transport characteristics of bacteria) By the classification method of the present invention, the characteristics of the following bacteria are determined. 〇E.coli (Classification result 1) Na + in the presence of 3 the accumulation rate of H-L-Ala is higher than that of H-L-Ala in the absence of Na + in the absence of 3 and is comparable to the accumulation rate of H-L-Met in the absence of Na + in the presence of 3 the accumulation rate of H-L-Met is + in the absence of 3 comparable to the accumulation rate of H-L-Met in the absence of Na (Classification result 2) Na + in the presence of 3 in the inhibition of H-L-Ala accumulation, L-Ala inhibition is higher than D-Ala inhibition, and + in the absence of 3 L-Ala inhibition and D-Ala inhibition are comparable in the inhibition of H-L-Ala accumulation, and + in the presence of 3 in the inhibition of H-L-Met accumulation, L-Met inhibition is higher than D-Met inhibition, and + in the absence of 3 L-Met inhibition is higher than D-Met inhibition in the inhibition of H-L-Met accumulation

[0031] (Classification results of amino acid transport characteristics of cancer cells) The classification method of the present invention determines the following cell characteristics 〇Lung cancer cells (Classification result 1) Na + in the presence of 3 the accumulation rate of H-L-Ala is + in the absence of 3 higher than that of H-L-Ala in the absence of Na, and + in the presence of 3 the accumulation rate of H-L-Met is + in the absence of 3 higher than that of H-L-Met in the absence of Na (Classification result 2) Na + in the presence of 3In the inhibition of H-L-Ala accumulation, BCH inhibition is higher than MeAIB inhibition, and Na + in the absence of 3 In the inhibition of H-L-Ala accumulation, BCH inhibition is observed but MeAIB inhibition is not, and Na + in the presence of 3 In the inhibition of H-L-Met accumulation, BCH inhibition is observed but MeAIB inhibition is not, and Na + in the absence of 3 In the inhibition of H-L-Met accumulation, BCH inhibition is observed but MeAIB inhibition is not.

[0032] 〇Glioblastoma multiforme cells (Classification result 1) Na + in the presence of 3 The accumulation rate of H-L-Ala is higher than that of H-L-Ala in the absence of Na + in the absence of 3 and is comparable to the accumulation rate of H-L-Met in the absence of Na + in the presence of 3 The accumulation rate of H-L-Met is + in the absence of 3 at the same level as the accumulation rate of H-L-Met. (Classification result 2) Na + in the presence of 3 In the inhibition of H-L-Ala accumulation, BCH inhibition is higher than MeAIB inhibition, and Na + in the absence of 3 In the inhibition of H-L-Ala accumulation, BCH inhibition is higher than MeAIB inhibition, and Na + in the presence of 3 In the inhibition of H-L-Met accumulation, there is no MeAIB inhibition, and Na + in the absence of 3 In the inhibition of H-L-Met accumulation, BCH inhibition is observed but MeAIB inhibition is not.

[0033] 〇Brain tumor cells (Classification result 1) Na + in the presence of 3 The accumulation rate of H-L-Ala is + in the absence of3 Higher than the accumulation rate of H-L-Ala and, in the presence of Na + in the presence of 3 the accumulation rate of H-L-Met is higher than that of H-L-Met + in the absence of 3 in the absence of Na. (Classification result 2) Na + in the presence of 3 in the inhibition of H-L-Ala accumulation, BCH inhibition and MeAIB inhibition are of the same degree, and in the absence of Na + in the absence of 3 in the inhibition of H-L-Ala accumulation, BCH inhibition is observed but MeAIB inhibition is not, and in the presence of Na + in the presence of 3 in the inhibition of H-L-Met accumulation, MeAIB inhibition is slight, and in the absence of Na + in the absence of 3 in the inhibition of H-L-Met accumulation, BCH inhibition is observed but MeAIB inhibition is not.

[0034] (Patient-derived sample) In the present invention, the "patient" is not particularly limited, but preferably exemplified by humans and non-human mammals suspected of bacterial infection or cancer disease, and humans and non-human mammals diagnosed with cancer disease. In the present invention, the "sample" is not particularly limited, but refers to tissues, liquids, cells, and mixtures thereof isolated from a patient, and examples thereof include tumor biopsy, cerebrospinal fluid, intrapleural fluid, intraperitoneal fluid, lymph fluid, skin section, blood, urine, feces, sputum, respiratory organs, intestinal tract, urogenital organs, saliva, milk, digestive organs, and cells collected therefrom.

[0035] (Radioactive tracer) In each method of the present invention, a radioisotope may be added as a tracer, and the movement and distribution of the target substance may be traced by measuring its radioactivity. As the radioactive tracer, 18 F-2-deoxy-2-fluoro-D-glucose( 18 F-FDG) is currently used for cancer diagnosis (Non-Patent Documents 3 and 4).

[0036] (Growth phase of bacteria) The growth of bacteria is divided into four growth phases: the lag phase, the logarithmic growth phase, the stationary phase, and the death phase. The lag phase (log phase) is a state where the growth of bacteria almost stops, and it is a preparation period for bacteria to adapt to the new environment and start growing. In the logarithmic growth phase (log phase), the enzyme activity in the bacterial cells is the most active, and bacteria grow vigorously. In the stationary phase, the growth rate of bacteria decreases due to the lack of nutrients and the accumulation of harmful metabolites, and the death of bacteria begins. In the death phase (phase of decline), the number of viable bacteria decreases and the number of dead bacteria increases (Non-Patent Documents 2 and 5).

[0037] (Amino acid metabolism of bacteria) For bacteria to grow and multiply, it is essential to take in necessary nutrients from the outside world and use these nutrients to produce substances necessary for the synthesis of cell components and the acquisition of energy. The nutrients necessary for the growth and multiplication of bacteria are the same as those of higher organisms, including water, carbon sources, nitrogen sources, inorganic salts, growth factors, etc. (Non-Patent Document 2). Amino acids are essential for the maintenance of bacterial life as a nitrogen source, and amino acid transport functions are ubiquitous in bacterial cells (Non-Patent Document 6). Bacteria directly take in external amino acids into the cells and efficiently carry out protein synthesis, carbon source acquisition, and nitrogen metabolism necessary for growth and multiplication. In addition, bacteria take in various amino acids such as methionine, lysine, tyrosine, and histidine (Non-Patent Documents 7 to 10).

[0038] (Bacterial infectious diseases) As used herein, the term "bacterial infectious disease" refers to an infectious disease caused by the growth of bacteria in a living body. The bacteria are not particularly limited, and examples include enteropathogenic Escherichia coli (EPEC), enteroinvasive Escherichia coli (EIEC), enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), enteroaggregative Escherichia coli (EAEC), Clostridium perfringens, Vibrio cholerae O1 and Vibrio cholerae O139, Corynebacterium diphtheriae, non-tuberculous mycobacteria (NTM), Methicillin Resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Legionella spp., Serratia marcescens, Streptococcus pyogenes, and the like. Bacterial infectious diseases are not particularly limited, and examples include diarrheogenic Escherichia coli infection, Clostridium perfringens infection, cholera, diphtheria, nontuberculous mycobacteriosis, MRSA infection, Pseudomonas aeruginosa infection, Legionella pneumonia, Serratia infection, severe group A streptococcal infection, and the like.

[0039] (Diagnostic methods for bacterial infectious diseases) In various methods using the radioisotope-labeled amino acids of the present invention, the accumulation of the amino acids in bacteria (e.g., cells infected with the pathogen of a bacterial infectious disease) and cancer cells is detected. (1) Administering a radioisotope-labeled amino acid to a subject (patient); (2) Detecting the administered amino acid; and (3) Determining the characteristics of amino acid transport, and determining the presence or progression of a bacterial infection or cancer from the detection results.

[0040] Regarding (1), the method of administering the radioisotope-labeled amino acid can be administered in the same manner as generally known radiological diagnostic agents. The administration method is not particularly limited, and examples include intravenous injection, subcutaneous injection, intradermal injection, and intramuscular injection. The timing of administration can be appropriately determined according to the condition of the subject (patient) and the treatment or diagnosis situation.

[0041] Regarding (2), the method of detecting the administered radioisotope-labeled amino acid is not particularly limited, and for example, it can be performed by imaging using a known method. For example, by detecting the radiation emitted from the compound using nuclear medicine images such as PET or SPECT, imaging of a bacterial infection (bacterial growth in the patient's body) is possible.

[0042] (Radioactivity) The radioactivity of the radioisotope-labeled amino acid of the present invention can be appropriately set to a radioactivity detectable in the step of (2). For example, in the case of PET imaging, it is necessary to have a radioactivity that enables PET imaging. For example, for the purpose of performing PET imaging on an adult, it is sufficient to have a radioactivity of 50 to 225 MBq at the time of use.

[0043] Regarding (3), the method for determining the characteristics of amino acid transport, and for determining the presence or progression of bacterial infections or cancer from the detection results is not particularly limited. For example, the signal intensity and / or signal distribution of radioactivity detected in the subject is compared with the signal intensity and / or signal distribution of radioactivity detected at the same site in a comparison subject known not to have a bacterial infection (a comparison subject negative for bacterial infection) to which a radioisotope-labeled amino acid has been administered, or with the signal intensity and / or signal distribution of radioactivity detected at the same site in a comparison subject known to have a bacterial infection (a comparison subject positive for bacterial infection) to which a radioisotope-labeled amino acid has been administered, and determination can be made thereby.

Example

[0044] Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited by these examples in any way.

Example

[0045] [Na + -dependent membrane transport activity evaluation] Escherichia coli K-12 strain was cultured with the tracer labeled amino acids + H-L-Ala or 3 H-L-Met in a medium in the presence of sodium (Na 3 -PBS) and a medium without sodium (Ch-PBS), and the accumulation amount of amino acids and the contribution rate of the sodium-dependent transport system were evaluated.

[0046] (Bacterial strain used and preculture conditions) In this example, a strain derived from E. coli K-12 was used as a model bacterium. As the medium for preculture, THY medium obtained by adding 0.2% Yeast extract (Becton, Dickinson and Company) to Todd’s Hewitt Broth (THB medium; Becton, Dickinson and Company) was used. The preculture conditions were 37°C and 5% CO2 for 14 hours or more.

[0047] (Cells and Pre-culture Conditions) In this example, DU145 (prostate cancer) and T98G (brain tumor) were used as cells. As the pre-culture medium, for DU145, RPMI1640 Medium mixed with 10% FBS and 1% sodium pyruvate was used. For T98G, Eagle's Minimum Essential Medium (Wako) mixed with 10% FBS and 1% sodium pyruvate was used. The culture conditions were 37°C under aerobic conditions, and confluent cultures were obtained on a 24-well plate.

[0048] (Contribution Rate Calculation Experiment of Na + -Dependent Amino Acid Transport System) The E. coli K-12-derived strain pre-cultured by the above method was added with 800 μL of the bacterial solution to a 50 mL conical tube containing 20 mL of amino acid-free D-MEM, and shaken and cultured at 160 rpm and 37°C for 6 hours. After that, D-MEM was replaced with PBS containing Na + (Na + -PBS) and PBS with Na + replaced by choline (Ch-PBS), each with 20 mL. Then, 900 μL of the bacterial solution was aliquoted into 1.5 mL Eppendorf tubes. After pre-incubation at 37°C for 10 minutes, 7.4 kBq of 3 H-L-Ala and 3 H-L-Met, the tracers, were administered to each tube and taken up for 5 minutes while gently shaking in a warm bath at 37°C. After that, centrifugation was performed at 7000 G and 4°C for 6 minutes, the supernatant was removed, and when the pellet was resuspended, 1 mL of Na + -PBS and Ch-PBS were added, and the centrifugation operation was performed twice for washing. After washing, the supernatant was removed, the pellet was resuspended, 1 mL of 0.1 M NaOH was added to lyse the E. coli, and a sample lysate was prepared. 500 μL was aliquoted from the sample lysate, 4 mL of a liquid scintillation cocktail was added, and the radioactivity was measured using a liquid scintillation counter.

[0049] (Experiment for calculating the contribution rate of the Na + -dependent transport system in tumor cells) For DU145 cells or T98G cells pre-cultured on a 24-well plate by the above method, the pre-culture medium in each well was removed and replaced with 300 μL of either Na + -PBS or Ch-PBS. After pre-incubating at 37 °C for 10 minutes, the tracer 3 H-L-Ala or 3 H-L-Met was administered at 9.25 kBq to each well and taken up for 5 minutes while gently shaking in a 37 °C water bath. After 5 minutes, the solution in the well was removed, and the cells and the well were washed twice with either Na + -PBS or Ch-PBS. 500 μL of 0.1 M NaOH was added to each well to lyse the cells. 350 μL of the cell lysate was mixed with a liquid scintillation cocktail, and the radioactivity was measured using a liquid scintillation counter.

[0050] (Evaluation of the contribution rate of the Na + -dependent transport system) "Contribution rate of the Na + -dependent transport system of labeled amino acids in the calculation experiment". The accumulation rate of the labeled amino acid calculated from the radioactivity measured in the "calculation experiment of the contribution rate of the Na + -dependent transport system of labeled amino acids" was divided by the accumulation rate of the labeled amino acid in Escherichia coli K-12 strain when using Na + -PBS, and multiplied by 100 to calculate the value (%) of Ch-PBS relative to 100% of Na-PBS. The difference (%) between the two values was shown in Table 1 as the contribution rate of the Na Also, for the "Na +In the accumulation rate of labeled amino acids calculated from the radioactivity measured in the "experiment for calculating the contribution rate of the Na-dependent transport system", the accumulation rate of labeled amino acids in each tumor cell when using Ch-PBS was divided by the accumulation rate of labeled amino acids in each tumor cell when using Na + -PBS, and the value (%) of Ch-PBS relative to 100% of Na-PBS was calculated by multiplying by 100. The difference (%) between the two values was shown in Table 1 as the contribution rate of the Na + -dependent transport system in tumor cells.

[0051] (Results) In Escherichia coli 3 The results of the accumulation changes of H-L-Ala or 3 H-L-Met are shown in Figure 1. From Figure 1, it was confirmed that in the Escherichia coli K-12 strain, the Na + -dependent transport system contributes to the transport of L-Ala, and the contribution rate is 13%. The contribution rates of the Na + -dependent transport systems in tumor cells and bacteria are shown in Table 1. From Table 1, it became clear that bacteria have a Na + -dependent amino acid transport system similar to that of human tumor cells. However, depending on the amino acid serving as the substrate, the contribution rate of the Na + -dependent transport system involved in accumulation is different between tumor cells and bacteria, so it was confirmed that bacteria have a different amino acid transport system from tumor cells.

[0052]

Table 1

Example

[0053] [Evaluation of the inhibitory effect of inhibitor loading on the accumulation of 3 H-L-Ala and 3 H-L-Met] System A (ATA1, ATA2, ATA3, etc.) co-transports substrates using the Na + concentration gradient inside and outside the cell, so Na +It is one of the dependent amino acid transporters (AAT). System L (LAT1, LAT2, LAT3, LAT4, etc.) transports substrates using the concentration gradient of substances other than Na + and is one of the Na + independent AAT. To investigate the characteristics of the accumulation mechanism involved in accumulation, α-methylaminoisobutyric acid (MeAIB), a specific inhibitor for the Na + dependent membrane transport system transporter of system A, and 2-amino-2-norbornane-carboxylic acid (BCH), a specific inhibitor for the Na + independent membrane transport system transporter of system L, were loaded as accumulation inhibitors, and the accumulation changes of the tracers [2,3- 3 H]-L-alanine( 3 H-L-Ala) or [S-methyl- 3 H]-L-methionine( 3 H-L-Met) were evaluated.

[0054] (Cells used and pre-culture conditions) In this example, H441 (lung adenocarcinoma), T98G (glioblastoma multiforme), and DU145 (prostate cancer) were used as cells. As the pre-culture medium, RPMI1640 Medium mixed with 10% FBS and 1% sodium pyruvate was used for H441 and DU145, respectively. For T98G, Eagle's Minimum Essential Medium mixed with 10% FBS and 1% sodium pyruvate was used. The culture conditions were 37°C under aerobic conditions, and the cells were made confluent on a 24-well plate. All experiments in this example used cells in this state.

[0055] (Accumulation inhibition experiment of Escherichia coli) A bacterial solution (800 μL) of an E. coli K-12-derived strain precultured in the same manner as in Example 1 was added to a 50-mL conical tube containing 20 mL of amino acid-free D-MEM, and cultured with shaking at 160 rpm at 37° C. for 6 hours. Then, D-MEM was diluted with Na + PBS containing (Na + -PBS) and Na + After replacing the PBS with 20 mL of choline-containing PBS (Ch-PBS), the bacterial solution was divided into 900 μL aliquots in 1.5 mL Eppendorf tubes. After preincubation at 37°C for 10 minutes, each tube was filled with tracer 3 HL-Ala or 3 HL-Met was administered at 7.4 kBq, and the inhibitor MeAIB or BCH was adjusted to a final concentration of 1 mM, and the cells were allowed to take up the inhibitor for 5 minutes while being gently shaken in a 37°C water bath. Thereafter, the mixture was centrifuged at 7000 G at 4° C. for 6 minutes, the supernatant was removed, and the pellet was loosened, to which 1 mL of Na-PBS and Ch-PBS was added, and the mixture was centrifuged twice to wash it. After washing, the supernatant was removed, the pellet was loosened, and 1 mL of 0.1 M NaOH was added to dissolve the E. coli, producing a sample solution. 500 μL of the sample solution was taken, 4 mL of liquid scintillation cocktail was added, and the radioactivity was measured using a liquid scintillation counter. Na + - PBS treatment group (Na + The control for + - The accumulation rate of the labeled amino acid taken up into the bacteria or cells when PBS was used as the medium and only the labeled amino acid was administered without loading with an inhibitor. For the control group (Ch) treated with Ch-PBS, Ch-PBS was used as the medium. Na + The control value is set as 100% and the values ​​at each inhibitor loading are 3 HL-Ala or 3 Accumulation rate of HL-Met was calculated as % of control / Na +It was evaluated by. Ch and Na + The contribution rate of amino acid transport of the Na + -dependent transport system can be known from the accumulation rate difference with.

[0056] (Tumor cell accumulation inhibition experiment) For H441 cells, T98G cells or DU145 cells pre-cultured in the same manner as the DU145 cells and T98G cells of Example 1, the pre-culture medium in each well was removed and replaced with 300 μL of Na + -PBS or Ch-PBS respectively. After pre-incubating at 37 °C for 10 minutes, the tracer 3 H-L-Ala or 3 H-L-Met was administered at 9.25 kBq, and it was taken up for 5 minutes while gently shaking in a warm bath at 37 °C. After 5 minutes, the solution in the well was removed, and the cells and the inside of the well were washed twice with Na + -PBS or Ch-PBS. 500 μL of 0.1 M NaOH was added to each well to lyse the cells, 350 μL of the cell lysate was mixed with a liquid scintillation cocktail, and the radioactivity was measured with a liquid scintillation counter.

[0057] (Results) 3 H-L-Ala or 3 The results of the accumulation changes of H-L-Met are shown in FIGS. 2 and 3. From FIG. 2, in H441, T98G and Du145, MeAIB, a specific inhibitor for system A, which is a Na + -dependent amino acid transport system, inhibited the accumulation of L-Ala in the presence of Na + . Since BCH also inhibited the accumulation of L-Ala in the presence of Na + , BCH is a Na + -independent system L specific inhibitor, but it was confirmed that it also inhibits the Na 0,+ , B 0 -containing Na + -dependent system. Also, as shown in Figure 2, the accumulation of L-Ala in Escherichia coli K-12 strain does not show the Na + dependency observed in tumor cells, and it was confirmed that there was no inhibitory effect with MeAIB, a specific inhibitor for system A of tumor cells. As shown in Figure 3, in H441, T98G, and Du145, BCH, a specific inhibitor for system L, a Na + independent amino acid transport system, inhibited the accumulation of L-Met in the absence of Na + . Since MeAIB also inhibited the accumulation of L-Met in the absence of Na + , it was confirmed that MeAIB is a specific inhibitor for Na + dependent system A, but also inhibits Na + independent systems including system PAT. Also, as shown in Figure 3, it was confirmed that the accumulation of L-Met in Escherichia coli K-12 strain was not inhibited by BCH, a specific inhibitor for system L of tumor cells.

Example

[0058] [Evaluation of the inhibitory effect on the accumulation of 3 H-L-Ala or 3 H-L-Met in Escherichia coli] Escherichia coli K-12 strain was cultured with the tracer labeled amino acid 3 H-L-Ala or 3 H-L-Met in a medium with sodium (Na-PBS) and a medium without sodium (Ch-PBS). For the tracer 3 H-L-Ala, the non-radioactive structural isomers L-alanine (L-Ala) or D-alanine (D-Ala) were used as accumulation inhibitors, and for the tracer 3 H-L-Met, the non-radioactive structural isomers L-methionine (L-Met) or D-methionine (D-Met) were used as accumulation inhibitors, and the inhibitory effect of the structural isomers on the accumulation amount of amino acids was evaluated when loaded.

[0059] (Accumulation inhibition experiment) 800 μL of the precultured E. coli K-12-derived strain obtained by the above method was added to a 50 mL conical tube containing 20 mL of amino acid-free D-MEM, and the mixture was cultured with shaking at 160 rpm and 37 °C for 6 hours. After that, D-MEM was replaced with PBS (Na + -PBS) containing + and PBS (Ch-PBS) in which + was replaced with choline, each to 20 mL. Then, 900 μL of the bacterial solution was aliquoted into 1.5 mL Eppendorf tubes. After pre-incubation at 37 °C for 10 minutes, 3 H-L-Ala or 3 H-L-Met, each at 7.4 kBq, as a tracer, and for the tracer 3 H-L-Ala, L-Ala or D-Ala, and for the tracer 3 H-L-Met, L-Met or D-Met were administered as inhibitors adjusted to a final concentration of 1 mM, and the mixture was allowed to take up for 5 minutes with gentle shaking in a warm bath at 37 °C. Thereafter, centrifugation was performed at 7000 G and 4 °C for 6 minutes, the supernatant was removed, and when the pellet was loosened, 1 mL of Na-PBS and Ch-PBS was added, and the centrifugation operation was repeated twice for washing. After completion of washing, the supernatant was removed, the pellet was loosened, 1 mL of 0.1 M NaOH was added to lyse the E. coli, and a sample lysate was prepared. 500 μL was aliquoted from the sample lysate, 4 mL of a liquid scintillation cocktail was added, and the radioactivity was measured with a liquid scintillation counter.

[0060] (Results) 3 The results of the accumulation changes of 3 H-L-Ala or H-L-Met are shown in Fig. 4. 3 From Fig. 4, in the accumulation of +There is a dependent L-form selective transport system, but its contribution is small, and both the L-form and D-form showed similar accumulation. On the other hand, in the 3 accumulation of H-L-Met in Escherichia coli K-12 strain, when L-Met or its optical isomer D-Met, which causes self-inhibition, or L-Ala or its optical isomer D-Ala is added, in the case of Met, Na + the contribution by the non-dependent L-form selective transport system was large, indicating that the L-form was preferentially taken up. From FIGS. 1 and 4, it was found that in Escherichia coli, Ala has lower optical isomer selectivity compared to Met, and Met has higher optical isomer selectivity than Ala. Generally, in human cancer cells and the like, the optical isomer selectivity is high like that of Met. Therefore, also in bacteria such as Escherichia coli, by utilizing these transport characteristics, it can provide useful information for the design strategy of pathogen imaging agents based on amino acids as the basic skeleton and can be used for the differential diagnosis of bacteria.

Example

[0061] [Evaluation of Sodium + Dependent Membrane Transport Activity Using Escherichia coli EC-14 Strain] The pathogen model Escherichia coli EC-14 strain was cultured with the tracer labeled amino acid + H-L-Ala or 3 H-L-Met in a medium with sodium (Na 3 -PBS) and a medium without sodium (Ch-PBS), and the accumulation amount of the amino acid and the contribution rate of the sodium-dependent transport system were evaluated based on the method described in Example 1. (Bacterial Strain Used and Preculture Conditions) In this example, a strain derived from E. coli EC-14 was used as the model bacterium. As the medium for pre-culture, THY medium prepared by adding 0.2% Yeast extract (Becton, Dickinson and Company) to Todd’s Hewitt Broth (THB medium; Becton, Dickinson and Company) was used. For the pre-culture conditions, the EC-14 strain was cultured in THY medium at 37 °C and 5% CO2 for 9 hours, and then a part of the culture solution was resuspended in THY medium for an additional 14 hours or more. (Cells used and pre-culture conditions) In this example, DU145 (prostate cancer) and T98G (brain tumor) were used as cells. As the pre-culture medium, RPMI1640 Medium mixed with 10% FBS and 1% sodium pyruvate was used for DU145. For T98G, Eagle's Minimum Essential Medium (Wako) mixed with 10% FBS and 1% sodium pyruvate was used. The culture conditions were 37 °C under aerobic conditions until confluent on a 24 well Plate. (Calculation experiment of the contribution rate of the Na + -dependent transport system) The E. coli EC-14-derived strain pre-cultured by the above method was added with 400 μL of the bacterial solution to a 50 mL conical tube containing 20 mL of amino acid-free D-MEM, and cultured with shaking at 160 rpm and 37 °C for 3 hours. After that, D-MEM was replaced with PBS containing Na + (Na + -PBS) and PBS with Na + replaced with choline (Ch-PBS), each with 20 mL. Then, 900 μL of the bacterial solution was aliquoted into 1.5 mL Eppendorf tubes. After pre-incubation at 37 °C for 10 minutes, 7.4 kBq of the tracers 3 H-L-Ala, 3 H-L-Met were administered to each tube and taken up for 5 minutes while gently shaking in a warm bath at 37 °C. Subsequently, it was centrifuged at 7000 G for 10 minutes at 4°C, the supernatant was removed, and when the pellet was loosened, 1 mL of Na + -PBS and Ch-PBS were added, and the centrifugation operation was performed twice for washing. After completion of washing, the supernatant was removed, the pellet was loosened, 1 mL of 0.1 M NaOH was added to lyse Escherichia coli, and a sample lysate was prepared. 500 μL was aliquoted from the sample lysate, 4 mL of a liquid scintillation cocktail was added, and the radioactivity was measured with a liquid scintillation counter.

[0062] (Results) In Escherichia coli EC-14 3 The results of the accumulation change of H-L-Ala or 3 H-L-Met are shown in Fig. 5. From Fig. 5, in the Escherichia coli EC-14 strain, it was confirmed that a Na + -dependent transport system contributes to the transport of L-Ala and L-Met. Note that the transport of L-Met in the Escherichia coli EC-14 strain is different from that of the Escherichia coli K-12 strain (reference: Fig. 1), and it was confirmed that a Na + -dependent transport system contributes. Thus, by detecting the difference in the transport of L-Met, it is possible to identify whether it is either the Escherichia coli EC-14 strain or the Escherichia coli K-12 strain.

Example

[0063] [Evaluation of the inhibitory effect on the accumulation of 3 H-L-Ala and 3 H-L-Met by inhibitor loading using Escherichia coli EC-14] Evaluation was carried out based on the methods described in Example 2 and Example 4 using the Escherichia coli EC-14 strain, which is a pathogen model. Specifically, the culture conditions adopted were those described in Example 4.

[0064] (Results) 3 The results of the accumulation change of H-L-Ala or 3 H-L-Met are shown in Figs. 6 and 7. As shown in Figure 6, the accumulation of L-Ala in Escherichia coli EC-14 strain was Na + -dependent, which was different from that in Escherichia coli K-12 strain (see Figure 2). Therefore, by detecting the difference in the transport of L-Ala, it is possible to identify whether it is Escherichia coli EC-14 strain or Escherichia coli K-12 strain. As shown in Figure 7, the accumulation of L-Met in Escherichia coli EC-14 strain showed the same result as that in Escherichia coli K-12 strain (see Figure 3).

Example

[0065] [Evaluation of the inhibitory effect on the accumulation of 3 H-L-Ala or 3 H-L-Met using Escherichia coli EC-14] Evaluation was carried out based on the methods described in Examples 3 and 4 using Escherichia coli EC-14 strain, which is a pathogen model. Specifically, the culture conditions adopted were those described in Example 4.

[0066] (Results) 3 The results of the change in the accumulation of 3 H-L-Ala or H-L-Met are shown in Figure 8. As shown in Figure 8, in the accumulation of 3 H-L-Ala in Escherichia coli EC-14 strain, when D-Ala was added in the absence of sodium, it was confirmed that it was higher compared to its accumulation in Escherichia coli K-12 strain (see Figure 4). Therefore, by detecting the difference in the transport of L-Ala with the addition of D-Ala in the absence of sodium, it is possible to identify whether it is Escherichia coli EC-14 strain or Escherichia coli K-12 strain. The accumulation of 3 H-L-Met in Escherichia coli EC-14 strain showed the same result as that in Escherichia coli K-12 strain (see Figure 4).

[0067] From the results of Figures 5 to 8, it was confirmed that there were the following differences in Escherichia coli EC-14 strain compared with Escherichia coli K-12 strain. Furthermore, by detecting this difference, it is possible to identify whether it is Escherichia coli EC-14 strain or Escherichia coli K-12 strain. (1) Accumulation amount of labeled L-Met in the presence of sodium compared to that in the absence of sodium (2) Accumulation amount of labeled L-Ala in the presence of sodium compared to that in the absence of sodium (3) Accumulation amount of labeled L-Ala when D-Ala is added in the absence of sodium Similar to the above examples, due to the difference in the inhibitory effect on the accumulation amount of the radioactive compound by pathogenic bacteria, it can be applied to the discrimination test of other bacterial species or strains of pathogenic bacteria.

Example

[0068] In this example, 125 L-MIT, an I-labeled tyrosine derivative ( 125 I-3-iodo-L-tyrosine), D-MIT ( 125 I-3-iodo-D-tyrosine), and L-AMT ( 125 I-3-iodo-alpha-methyl-L-tyrosine) accumulation in Escherichia coli was evaluated.

[0069] (Bacterial species used and pre-culture conditions) As the medium for pre-culture, THY medium obtained by adding 0.2% Yeast extract (Becton, Dickinson and Company) to Todd’s Hewitt Broth (THB medium; Becton, Dickinson and Company) was used. The pre-culture conditions were as follows: For the K-12 strain, it was cultured at 37 °C under 5% CO2 for 14 hours or more. For the EC-14 strain, after culturing at 37 °C and 5% CO2 for 9 hours using THY medium, a part of the culture solution was resuspended in THY medium and cultured for 14 hours or more. (Labeled amino acids used) For the labeling of L-Tyr, D-Tyr, and L-AMT 125I L-Tyr (Nacalai Tesque), D-Tyr (Peptide Institute), and L-AMT (Sigma-Aldrich) were selected as the labeling raw materials, and all other reagents were of special grade. For L / D-Tyr and L-AMT 125The I-labeling reaction was carried out under carrier-free conditions by a direct labeling method using chloramine-T as an oxidizing agent. Subsequently, separation and purification were performed using high-performance liquid chromatography (HPLC, Hitachi), and 125 I-L-MIT, 125 I-D-MIT and 125 I-L-AMT were obtained. (Labeling method) The labeling raw materials L / D-Tyr, L-AMT, and chloramine-T (Nacalai tesque) were each dissolved in 0.4 M and 0.05 M phosphate buffer (pH 6.2), and each was adjusted to 1×10 -7 mol / 100 μL and 1×10 -8 mol / 25 μL. To carrier-free 125 131I-NaI (5 MBq, PerkinElmer), 100 μL of the L / D-Tyr, L-AMT solution was added, and then 25 μL of the chloramine-T solution was added to initiate the reaction. Fifteen minutes after the start of the reaction, 25 μL of a solution obtained by diluting a saturated solution of sodium pyrosulfite (Wako Pure Chemical Industries) 10-fold with 0.05 M phosphate buffer (pH 6.2) was added to stop the reaction. The labeled 125 I-L-MIT, 125 I-D-MIT and 125 I-L-AMT were analyzed by silica gel thin layer chromatography (TLC, Merck), and the labeling rate was evaluated using an auto-well γ-counter (AccuFLEXγ7000, Aloka). Also, methanol (≥ 99.5%, Sigma-Aldrich): acetic acid (Nacalai tesque) = 100:1 was used as the developing solvent for TLC. (Separation and purification method) 125 I-L-MIT, 125 I-D-MIT and 125High-performance liquid chromatography (HPLC, Hitachi) was used for the isolation and purification of I-L-AMT. The chromatogram was analyzed by PowerChrom (eDAQ). A normal-phase column was used for hydrophilic interaction chromatography (HILIC, Cosmosil, Nacalaitesque). After isolation and purification, nitrogen reflux was performed, and the collected 125 I-L-MIT, 125 I-D-MIT and 125 acetonitrile (≥ 99.5%, Sigma-Aldrich) contained in I-L-AMT was removed. (Time-course accumulation experiment of labeled amino acids) The E. coli K-12-derived strain pre-cultured by the above method was added with 800 μL of the bacterial solution to a 50 mL conical tube containing 20 mL of amino acid-free D-MEM, and shaken at 160 rpm and 37 °C. For the E. coli EC-14-derived strain, 400 μL of the bacterial solution was added to a 50 mL conical tube containing 20 mL of amino acid-free D-MEM, and shaken at 160 rpm and 37 °C. Thereafter, for the E. coli K-12-derived strain, after culturing for 2, 6, and 12 hours, and for the E. coli EC-14-derived strain, after culturing for 1, 3, and 6 hours, 900 μL of the bacterial solution was aliquoted into 1.5 mL Eppendorf tubes, and the tracers 125 I-L-MIT, 125 I-D-MIT and 125 I-L-AMT were administered at 7.4 kBq and taken up for 5 minutes while gently shaking in a 37 °C water bath. Thereafter, centrifugation was performed at 7000 G and 4 °C for 10 minutes, the supernatant was removed, and when the pellet was loosened, 1 mL of phosphate buffered saline (PBS; pH 7.3) was added, and the centrifugation operation was performed twice for washing. After completion of washing, the supernatant was removed, the pellet was loosened, 1 mL of 0.1 M NaOH was added to lyse Escherichia coli, and a sample lysate was prepared. 500 μL was aliquoted from the sample lysate, and the radioactivity was measured with a gamma counter. (Inhibitors used) As inhibitors, in addition to L-Tyr and D-Tyr, 3-Iodo-L-tyrosine (L-MIT) and 3-Iodo-D-tyrosine (D-MIT) were used. (Inhibition experiment of labeled amino acid accumulation) 7.4 kBq of the labeled amino acid and each inhibitor prepared to a final concentration of 1 mM were administered, and the procedure was the same as in Example 3. (Accumulation inhibition experiment) 800 μL of the bacterial solution was added to a 50 mL conical tube containing 20 mL of amino acid-free D-MEM, and the pre-cultured E. coli K-12-derived strain was shake-cultured at 160 rpm and 37 °C for 6 hours by the above method. 800 μL of the bacterial solution was added to a 50 mL conical tube containing 20 mL of amino acid-free D-MEM, and the pre-cultured E. coli K-12-derived strain was shake-cultured at 160 rpm and 37 °C. Also, in the case of the E. coli EC-14-derived strain, 400 μL of the bacterial solution was added to a 50 mL conical tube containing 20 mL of amino acid-free D-MEM, and the strain was shake-cultured at 160 rpm and 37 °C. Thereafter, for the E. coli K-12-derived strain, after 2, 6, and 12 hours of culture, and for the E. coli EC-14-derived strain, after 1, 3, and 6 hours of culture, 900 μL of the bacterial solution was aliquoted into 1.5 mL Eppendorf tubes, and the tracers 125 I-D-MIT and 3 H-L-Tyr at 7.4 kBq, and L-Tyr, D-Tyr, L-MIT, and D-MIT adjusted to a final concentration of 1 mM as inhibitors were administered, and the mixture was taken up for 5 minutes with gentle shaking in a water bath at 37 °C. Thereafter, centrifugation was performed at 7000 G and 4 °C for 10 minutes, the supernatant was removed, and when the pellet was loosened, 1 mL of phosphate buffered saline (PBS; pH 7.3) was added, and the centrifugation operation was performed twice for washing. After completion of washing, the supernatant was removed, the pellet was loosened, 1 mL of 0.1 M NaOH was added to lyse Escherichia coli, and a sample lysate was prepared. 500 μL was aliquoted from the sample lysate, 3 Regarding the accumulation of H-L-Tyr, 3 mL of a liquid scintillation cocktail was added, and the radioactivity was measured with a liquid scintillation counter. 125 The accumulation of I-D-MIT was measured for radioactivity with an autowell γ counter.

[0070] From the results in Fig. 9, it was confirmed that in both Escherichia coli EC-14 strain and Escherichia coli K-12 strain, only D-MIT, which is a radioisotope-labeled form of D-amino acid rather than L-amino acid utilized by the living body, specifically accumulated. Also, D-MIT highly accumulated in pathogenic Escherichia coli EC-14 strain. From the results in Fig. 10, 125 I-D-MIT or 3 Regarding the inhibitory effect of each inhibitor on the accumulation of H-L-Tyr, there was no difference between Escherichia coli EC-14 strain and Escherichia coli K-12 strain. In addition, by the method for classifying the transport characteristics of the amino acids of the present invention, 3 Since the uptake of H-L-Tyr into the bacterial cells is not inhibited by D-amino acids in both Escherichia coli EC-14 strain and Escherichia coli K-12 strain, it is considered that L-amino acid-selective transport characteristics similar to those of human cells are involved. On the other hand, 125 Although the uptake of I-D-MIT into the bacterial cells is selective for D-amino acids in both Escherichia coli EC-14 strain and Escherichia coli K-12 strain, it is considered that transport characteristics partially overlapping with the transport of L-amino acids are involved. A transport system showing such transport characteristics is not observed in human cells, and the existence of a transport system specific to bacteria was confirmed. Similarly, by evaluating the transport characteristics in other pathogenic bacteria, it can be applied to the discrimination test of bacteria.

[0071] Based on the results in FIGS. 9 to 10, it was confirmed that by administering a radioisotope-labeled substance such as D-MIT, which shows pathogen accumulation different from that of human cells, to a living body and imaging it, it can be applied to the imaging diagnosis of pathogen-infected sites.

Industrial Applicability

[0072] A method for classifying bacteria or cells, a method for classifying the transport characteristics of amino acids, a method for assisting the diagnosis of bacterial infections, and a method for assisting the diagnosis of cancer can be provided.

Claims

**Claim 1** A method for classifying cells, comprising the following steps: introducing a radioisotope-labeled amino acid into cells in the presence or absence of Na+ to identify whether the uptake of the radioisotope-labeled amino acid into the cells is Na+-dependent or Na+-independent; wherein the classification method 1) Administering the radioisotope-labeled amino acid 3 H-L-Ala to a patient-derived sample that is the cell, in the presence or absence of Na + ; 3 ​ 2) Administering the radiolabeled amino acid 3 H-L-Met to the patient-derived sample that is the cell, in the presence or absence of + Na, and + in the presence or absence of the radiolabeled amino acid 3 H-L-Met, and 3) Na + in the presence of 3 the accumulation rate of H-L-Ala is higher than that of H-L-Ala + in the absence of 3 and, when the accumulation rate of H-L-Met in the presence of Na + in the presence of 3 is comparable to the accumulation rate of H-L-Met + in the absence of 3 a step of determining glioblastoma multiforme comprises a method for classifying cells for use in an auxiliary method for diagnosing glioblastoma multiforme. **Claim 2** A method for classifying cells, comprising the following steps: introducing a radioisotope-labeled amino acid into cells in the presence of an inhibitor against an amino acid transporter and in the presence or absence of Na+ to identify whether the uptake of the radioisotope-labeled amino acid into the cells is inhibited; wherein the classification method 1) Administer either the radioactive isotope-labeled amino acid 3 H-L-Ala or an inhibitor of the amino acid transporter, MeAIB or BCH, to a patient-derived sample that is the cell, in the presence or absence of Na + 3 ​​ 2) Administer either H-L-Met, which is the radioisotope-labeled amino acid, or MeAIB or BCH, which is an inhibitor for the amino acid transporter, to the patient-derived sample that is the cell, in the presence or absence of Na + 3 ​​ 3) Na + in the presence of 3 in the inhibition of the accumulation of H-L-Ala, BCH inhibition is higher than MeAIB inhibition, and in the absence of Na + in the absence of 3 in the inhibition of the accumulation of H-L-Ala, BCH inhibition is higher than MeAIB inhibition, and in the absence of Na + in the presence of 3 in the inhibition of the accumulation of H-L-Met, there is no MeAIB inhibition, and in the absence of Na + in the absence of 3 a step of determining glioblastoma multiforme when BCH inhibition is observed but MeAIB inhibition is not in the inhibition of the accumulation of H-L-Met comprises a method for classifying cells for use in an auxiliary method for diagnosing glioblastoma multiforme.

Images