WO2022075354A1

WO2022075354A1 - A method for obtaining an index for the diagnosis of alzheimer's disease (ad)

Info

Publication number: WO2022075354A1
Application number: PCT/JP2021/036950
Authority: WO
Inventors: Mitsuhiro Yanagida; Takayuki Teruya
Original assignee: kinawa Institute of Science and Technology Graduate University
Current assignee: kinawa Institute of Science and Technology Graduate University
Priority date: 2020-10-06
Filing date: 2021-10-06
Publication date: 2022-04-14
Anticipated expiration: 2023-04-06
Also published as: JP2023549028A

Abstract

It is an object of the present invention to provide a novel method capable of simply and accurately obtaining an index for the diagnosis of Alzheimer's disease (AD). The present invention is related to a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the specific blood metabolite. The present invention is also related to a method for assisting in the diagnosis of AD by measuring the specific blood metabolite.

Description

A METHOD FOR OBTAINING AN INDEX FOR THE DIAGNOSIS OF ALZHEIMER'S DISEASE (AD)

The present invention relates to a method for obtaining an index for the diagnosis of Alzheimer’s disease (AD), an apparatus for obtaining an index for the diagnosis of AD, a system for obtaining an index for the diagnosis of AD, a kit for obtaining an index for the diagnosis of AD and a method of evaluating substances effective in improving AD.

Human blood metabolites have been well investigated to determine their abundance and biological significance, and for their potential use as diagnostic markers. For medical diagnosis, non-cellular metabolites from plasma or serum, are mostly commonly employed due to the simplicity in collecting and examining them. While mature human red blood cells (RBCs) lack nuclei and cellular organelles (NPL 1), RBCs utilize glycolysis for ATP production, maintain redox homeostasis, and osmoregulate (NPL 2). Their active metabolism supports cellular homeostasis and ensures lifespans of ~4 months (NPL 3). Their metabolites may reflect health status or environmental stresses differently than do metabolites of plasma. As RBCs occupy about half the total blood volume (ca. 5 L), their metabolite profiles, which have scarcely been investigated, seemed worthy of investigation.

Metabolomics is a branch of chemical biology that profiles metabolites in cells and organisms, using techniques such as liquid chromatography (LC)-mass spectrometry (MS). It usually deals with molecules <1.5 kDa, and is an important tool for studying metabolic regulation in combination with other comprehensive analyses, such as proteomics and transcriptomics. Recently we reported that among 133 compounds identified in human blood, 101 are also found in the fission yeast, Schizosaccharomyces pombe (NPL 4), implying that many metabolites might be evolutionarily conserved. Quantitative measurements of an array of compounds among individuals, offer profound insights into health or disease conditions and effects of nutrition, drugs, and stress. Moreover, comprehensive information about individual variation in metabolites could impact the future of medical science (NPL 5-11).

Alzheimer’s disease (AD) is a slowly progressive neurodegenerative disease and the principal cause of dementia characterized by profound forgetfulness (NPL 12-13). The cause of Alzheimer's disease is poorly understood (NPL 14-15). Mental and physical exercise and avoiding obesity may decrease the risk of AD (NPL 16). No medications or supplements have been definitively shown to decrease risk (NPL 17-18). It most often begins in people over 65 years of age, and about 6% of seniors are afflicted with it. In 2015, there were approximately 29.8 million people worldwide with AD, making it one of the most costly diseases in developed countries.

Rapoport SM, Schewe T, & Thiele B-J (1990) Maturational breakdown of mitochondria and other organelles in reticulocytes. in Erythroid Cells, ed Harris JR (Springer US), pp 151-194. van Wijk R & van Solinge WW (2005) The energy-less red blood cell is lost: erythrocyte enzyme abnormalities of glycolysis. Blood 106(13):4034-4042. Bax BE, Bain MD, Talbot PJ, Parker-Williams EJ, & Chalmers RA (1999) Survival of human carrier erythrocytes in vivo. Clin Sci (Lond) 96(2):171-178. Chaleckis R, et al. (2014) Unexpected similarities between the Schizosaccharomyces and human blood metabolomes, and novel human metabolites. Molecular BioSystems 10(10):2538. Fernie AR, Trethewey RN, Krotzky AJ, & Willmitzer L (2004) Metabolite profiling: from diagnostics to systems biology. Nat Rev Mol Cell Biol 5(9):763-769. Goodacre R, Vaidyanathan S, Dunn WB, Harrigan GG, & Kell DB (2004) Metabolomics by numbers: acquiring and understanding global metabolite data. Trends Biotechnol 22(5):245-252. Hirai MY, et al. (2004) Integration of transcriptomics and metabolomics for understanding of global responses to nutritional stresses in Arabidopsis thaliana. Proc Natl Acad Sci U S A 101(27):10205-10210. Kell DB (2004) Metabolomics and systems biology: making sense of the soup. Curr Opin Microbiol 7(3):296-307. Nicholson JK & Lindon JC (2008) Systems biology: Metabonomics. Nature 455(7216):1054-1056. Patti GJ, Yanes O, & Siuzdak G (2012) Innovation: Metabolomics: the apogee of the omics trilogy. Nat Rev Mol Cell Biol 13(4):263-269. Ramautar R, Berger R, van der Greef J, & Hankemeier T (2013) Human metabolomics: strategies to understand biology. Curr Opin Chem Biol 17(5):841-846. Crews, L., and Masliah, E. (2010). Molecular mechanisms of neurodegeneration in Alzheimer's disease. Hum Mol Genet 19, R12-20 Lane, C.A., Hardy, J., and Schott, J.M. (2018). Alzheimer's disease. Eur J Neurol 25, 59-70 Ballard, C., Gauthier, S., Corbett, A., Brayne, C., Aarsland, D., and Jones, E. (2011). Alzheimer's disease. The Lancet 377, 1019-1031 Burns, A., and Iliffe, S. (2009). Alzheimer's disease. BMJ 338, b158 Pope, S.K., Shue, V.M., and Beck, C. (2003). Will a healthy lifestyle help prevent Alzheimer's disease? Annu Rev Public Health 24, 111-132 Cummings, J., Aisen, P.S., DuBois, B., Frolich, L., Jack, C.R., Jr., Jones, R.W., Morris, J.C., Raskin, J., Dowsett, S.A., and Scheltens, P. (2016). Drug development in Alzheimer's disease: the path to 2025. Alzheimers Res Ther 8, 39 Hsu, D., and Marshall, G.A. (2017). Primary and Secondary Prevention Trials in Alzheimer Disease: Looking Back, Moving Forward. Curr Alzheimer Res 14, 426-440

It is an object of the present invention to provide a novel method capable of simply and accurately obtaining an index for the diagnosis of Alzheimer’s disease (AD). In addition, it is also an object of the present invention to provide a novel method capable of simply and accurately assisting in the diagnosis of AD.

Metabolites present in human blood document individual physiological states influenced by genetic, epigenetic, and life-style factors. In this study, we conducted non-targeted comprehensive analysis of blood metabolites in AD. Thorough metabolomics can supply complete information about metabolite abundance in each subject. In targeted analysis, metabolites to be analyzed are pre-determined; however, changes in abundance of untargeted (unexpected) metabolites are overlooked. While non-targeted analysis is far more laborious, the effort expended in this “no assumptions” approach is often recompensed by identification of crucial compounds overlooked by targeted analysis. A wealth of metabolite information can provide clues for understanding detailed metabolic changes occurring in AD.

Using liquid chromatography - mass spectroscopy (LC-MS), we conducted metabolomic analysis of Alzheimer’s disease (AD) using whole blood of AD patients in an attempt to identify new metabolic compounds related to AD, particularly in blood cells. Most blood metabolomics employed plasma. Metabolomics of blood cells have scarcely been investigated, particularly in relation to diseases, despite the fact that red blood cells (RBCs) account for about 40% of all blood metabolites (Chaleckis et al., 2016; Kameda et al., 2020; Teruya et al., 2019). In the present study, we identified AD-linked markers and performed principal component analysis (PCA), correlation and heatmap analysis using quantitative information of 33 metabolites identified. We show five groups of AD-related metabolites that seem to diagnose distinct AD symptoms.

In this work, we present a novel method for obtaining an index for the diagnosis of Alzheimer’s disease (AD). We also present a novel method for assisting in the diagnosis of AD in a subject, comprising measuring the amount of a specific blood metabolite in the subject.

The present inventions are as follows.
[1] A method for obtaining an index for the diagnosis of Alzheimer’s disease (AD) by measuring at least one blood metabolite selected from the group consisting of trimethyl-tryptophan, S-methyl-ergothioneine, betaine, indoxyl-sulfate, pantothenate, trimethyl-histidine, dimethyl-xanthine, ATP, methionine, kynurenine, trimethyl-tyrosine, trimethyl-phenylalanine, NADP⁺, 2-hydroxybutyrate, keto(iso)leucine, glycerophosphocholine, gluconate, pseudouridine, N6-acetyl-lysine, and dimethyl-guanosine.
[2] The method for obtaining an index for the diagnosis of Alzheimer’s disease (AD) according to [1], wherein the blood metabolite is trimethyl-tryptophan.
[3] The method for obtaining an index for the diagnosis of Alzheimer’s disease (AD) according to [1], wherein the blood metabolite is trimethyl-tryptophan and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, indoxyl-sulfate, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.
[4] The method for obtaining an index for the diagnosis of Alzheimer’s disease (AD) according to [1], wherein the blood metabolite is selected from the group of antioxidative aromatic compounds with trimethyl-ammonium consisting of S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, and trimethyl-tyrosine.
[5] The method for obtaining an index for the diagnosis of Alzheimer’s disease (AD) according to [1], wherein the blood metabolite is selected from the group of neurotoxic compounds consisting of indoxyl-sulfate, kynurenine, and N6-acetyl-lysine.
[6] The method for obtaining an index for the diagnosis of Alzheimer’s disease (AD) according to [1], wherein the blood metabolite is selected from the group of RBC enriched compounds consisting of ATP, NADP⁺, pantothenate, and gluconate.
[7] A method for assisting in the diagnosis of AD in a subject by measuring at least one blood metabolite selected from the group consisting of trimethyl-tryptophan, S-methyl-ergothioneine, betaine, indoxyl-sulfate, pantothenate, trimethyl-histidine, dimethyl-xanthine, ATP, methionine, kynurenine, trimethyl-tyrosine, trimethyl-phenylalanine, NADP⁺, 2-hydroxybutyrate, keto(iso)leucine, glycerophosphocholine, gluconate, pseudouridine, N6-acetyl-lysine, and dimethyl-guanosine.
[8] The method for assisting in the diagnosis of AD in a subject according to [7], wherein the blood metabolite is trimethyl-tryptophan.
[9] The method for assisting in the diagnosis of AD in a subject according to [7], wherein the blood metabolite is trimethyl-tryptophan and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, indoxyl-sulfate, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.
[10] The method for assisting in the diagnosis of AD in a subject according to [7], wherein the blood metabolite is selected from the group of antioxidative aromatic compounds with trimethyl-ammonium consisting of S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, and trimethyl-tyrosine.
[11] The method for assisting in the diagnosis of AD in a subject according to [7], wherein the blood metabolite is selected from the group of neurotoxic compounds consisting of indoxyl-sulfate, kynurenine, and N6-acetyl-lysine.
[12] The method for assisting in the diagnosis of AD in a subject according to [7], wherein the blood metabolite is selected from the group of RBC enriched compounds consisting of ATP, NADP⁺, pantothenate, and gluconate.
[13] An apparatus for obtaining an index for the diagnosis of Alzheimer’s disease (AD) in which an index for the diagnosis of Alzheimer’s disease (AD) is obtained by the method according to any one of [1] to [6].
[14] A system for obtaining an index for the diagnosis of Alzheimer’s disease (AD) in which AD is evaluated by the method according to any one of [1] to [6] or the apparatus according to claim [13].
[15] An apparatus for assisting in the diagnosis of AD in a subject in which the diagnosis of AD is assisted by the method according to any one of claims [7] to [12].
[16] A system for assisting in the diagnosis of AD in a subject in which the diagnosis of AD is assisted by the method according to any one of claims [1] to [6] or the apparatus according to [15].
[17] A method of evaluating substances which improve Alzheimer’s disease (AD) comprising the step of measuring a blood metabolite such as trimethyl-tryptophan, S-methyl-ergothioneine, betaine, indoxyl-sulfate, pantothenate, trimethyl-histidine, dimethyl-xanthine, ATP, methionine, kynurenine, trimethyl-tyrosine, trimethyl-phenylalanine, NADP⁺, 2-hydroxybutyrate, keto(iso)leucine, glycerophosphocholine, gluconate, pseudouridine, N6-acetyl-lysine, or dimethyl-guanosine as AD markers.
[18] A kit for evaluation of Alzheimer’s disease (AD) in which AD is evaluated by the method according to any one of claims [1] to [12] comprising blood collection tubes and blood metabolite compounds as detection standard.

Using comprehensive metabolomics, we identified 5 groups of metabolites (A-E), 20 of which are novel, possibly useful for diagnosis and therapy of forms of dementia, such as Alzheimer's disease (AD). Seven compounds of Group A may act as neurotoxins, whereas compounds of Group B-E may protect the CNS (Central Nerve System) against oxidative stress, maintain energy reserves, supply nutrients and neuroprotective factors. Interventions for Alzheimer's disease metabolomic markers may be accomplished either by inhibiting Group A compounds or by supplementing Group B-E compounds in patients.

The present invention based on these findings provides a novel method capable of simple and accurate diagnosis of Alzheimer's disease (AD). The present invention also provides a novel method for obtaining an index for the diagnosis of Alzheimer’s disease (AD). The present invention also provides a novel method for assisting in the diagnosis of AD in a subject, comprising measuring the amount of a specific blood metabolite in the subject. The 33 blood metabolites that we found are specifically increased or decreased in patients with Alzheimer's disease (AD). Therefore, using these blood metabolites as indicators, it is possible to distinguish Alzheimer's disease from frailty.

Subjects of Alzheimer’s disease (AD), healthy elderly (HE) and healthy young (HY) volunteers; BMI: body mass index. Blood samples of AD subjects (75~88 yr) were drawn at the National Ryukyu Hospital. Healthy subject (HE, 67~80 yr, and HY, 28~34 yr) samples were collected from volunteers at the Onna Clinic in Onna-son, Okinawa. Dot plot profiles of 33 AD-related metabolites.; (Fig.2-1 to 2-13) 26 compounds decreased in AD whole blood. Dot plot profiles are shown for ergothioneine, glutathione disulfide, betaine, ATP, glutamine, phenylalanine, trimethyl-tryptophan, tryptophan, glycerophosphocholine, dimethyl-xanthine, tyrosine, caffeine, methionine, histidine, gluconate, trimethyl-phenylalanine, S-methyl-ergothioneine, trimethyl-tyrosine, keto(iso)leucine, pantothenate, 2-hyroxybutyrate, dodecanoyl-carnitine, trimethyl-histidine, NADP+, uridine, and S-adenosyl-methionine in 24 subjects AD, HE, and HY. (Fig.2-14 to 2-17) Seven compounds increased in AD. Dot plot profiles for seven compounds (four nucleosides and three amino acid derivatives) that increased in AD patients compared with HE are shown. Peak areas were maximal for indoxyl sulfate and minimal for quinolinic acid. In AD patients various trimethylated compounds abundance decreased in blood; Three trimethylated compounds (betaine, glycerophosphocholine, dodecanoyl-carnitine) are synthesized in human body, whilst six other compounds (ergothioneine, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-phenylalanine, trimethyl-tryptophan, trimethyl-tyrosine) were derived dietary metabolites. These nine compounds decreased in whole blood of AD patients. PCA (principal component analysis) of AD, HE and HY subjects’ blood compounds; PCA of 33 AD or 6 selected AD compounds showed significant differences between AD and HE. PCA (principal component analysis) of AD, HE and HY subjects’ blood compounds were subjected. (Fig.4-1) A PCA plot using abundances of 33 (7 increased and 26 decreased) AD-related compounds. AD and HE, HY subjects were separated into two domains (see text). Black triangle, AD; white triangles, HE. (Fig.4-2) PCA analysis was performed using selected 6 AD markers (dimethyl-guanosine, pseudouridine, S-methyl-ergothioneine, ergothioneine, trimethyl-histidine, and NADP⁺). Figure 5 is divided into 4 parts (Fig.5-1 to 5-4). Correlation analysis of 33 AD markers; The correlation values r larger than +0.5 (dark grey) indicates a high correlation between the compounds, whilst r below -0.5 (light grey) was indicates a low correlation between the compounds. Compounds increased or decreased in AD patients were indicated by the arrows. Figure 6 is divided into 2 parts (Fig.6-1 and 6-2). Construction of heatmap using abundance data of 33 compounds for each subject; Standardized scores (T-scores) are represented by colors. The average value, white; the values above average, light grey to dark grey without “X”; the values below average, light grey to dark grey with “X”. Seven compounds that increased in AD (subject #1~8) tended to be light grey to dark grey without “X” in AD whereas tended to be light grey to dark grey with “X” in HE (#9~18). For remaining 26 compounds that decreased in AD, the result was opposite in AD and HE. Five subclasses of compounds that may underpin anti-AD activities

Before the present invention is described in detail, it is to be understood that this invention is not limited to the particular methodology, apparatuses, and systems described, as such methodology, apparatuses and systems can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

Unless defined otherwise or the context clearly dictates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described.

All publications mentioned herein are hereby incorporated by reference for the purpose of disclosing and describing the particular materials and methodologies for which the reference was cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Definitions
The term "blood metabolite" is used herein to refer to a low molecular compound involved in biological metabolic activity contained in blood constituents.

It is understood that aspects and embodiments of the invention described herein include "consisting" and/or "consisting essentially” of aspects and embodiments.

Other objects, advantages and features of the present invention will become apparent from the following specification taken in conjunction with the accompanying drawings.

A method for obtaining an index for the diagnosis of Alzheimer’s disease (AD)
The present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject. According to the present invention, by measuring the amount of a specific blood metabolite, it is possible to diagnose or assist in diagnosing whether a subject has AD or is at risk for AD.

Here, the sample used for measuring the amount of a specific blood metabolite in a subject may be at least one kind selected from the group consisting of whole blood, erythrocyte and plasma. It is preferable to use either whole blood or erythrocyte. It is more preferable to use any two of whole blood, erythrocyte and plasma. It is most preferable to use all of whole blood, erythrocyte and plasma as a sample.

As the blood metabolite in the present invention, it is preferable that the compound has a large difference in blood content between the AD patients group (AD) and healthy elderly (HE) subjects. We found 33 blood metabolites with significantly altered concentrations in blood of AD patients. These 33 blood metabolites can be used as indicators or biomarkers for AD.

The following 26 metabolites are significantly declined in AD patients compare to HE. Ergothioneine, glutathione disulfide, betaine and ATP were found to be most abundant metabolites that decreased in AD. Two other ergothioneine-related, but less abundant compounds, S-methyl-ergothioneine and trimethyl-histidine (hercynine), also declined in AD. Trimethyl-tryptophan (hypaphorine), trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, and trimethyl-tyrosine, all of which contain trimethylammonium ions also declined. Caffeine and its derivative, dimethyl-xanthine, uridine (a pyrimidine nucleoside), S-adenosyl-methionine (methyl donor), NADP⁺ (oxidoreductive coenzyme), pantothenate (vitamin B5), keto(iso)leucine (keto-acid), 2-hydroxybutyrate (lipid-degradation product) and gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, and tryptophan diminished in AD subjects. Among them, the degree of reduction for caffeine and dimethyl-xanthine in AD was quite significant (ratio, 0.04 ~ 0.09). Trimethyl-tryptophan (0.10) and trimethyl-tyrosine (0.08) were also strikingly declined. Therefore when the content of these compounds in the subject’s blood sample is lower than standard, the subject is diagnosed as having AD or is at high risk for AD.

The following 7 metabolites, comprising 3 nucleosides and 4 amino acid derivatives, increased in AD patients compare to HE. Those are indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine. Therefore when the content of these compounds in the subject’s blood sample is higher than standard, the subject is diagnosed as having AD or is at high risk for AD.

In the present invention, the blood metabolite comprises at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

Preferably, the blood metabolite comprises at least one metabolite selected from the group consisting of trimethyl-tryptophan, S-methyl-ergothioneine, betaine, indoxyl-sulfate, pantothenate, trimethyl-histidine, dimethyl-xanthine, ATP, methionine, kynurenine, trimethyl-tyrosine, trimethyl-phenylalanine, NADP⁺, 2-hydroxybutyrate, keto(iso)leucine, glycerophosphocholine, gluconate, pseudouridine, N6-acetyl-lysine, dimethyl-guanosine.

More preferably, the blood metabolite comprises at least one metabolite selected from the group consisting of trimethyl-tryptophan, S-methyl-ergothioneine, betaine, indoxyl-sulfate, pantothenate, trimethyl-histidine, dimethyl-xanthine, ATP, methionine, kynurenine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises at least one metabolite selected from the group of antioxidative aromatic compounds with trimethyl-ammonium consisting of ergothioneine, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, and trimethyl-tyrosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises at least one metabolite selected from the group of antioxidative aromatic compounds with trimethyl-ammonium consisting of S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, and trimethyl-tyrosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises at least one metabolite selected from the group of neurotoxic compounds consisting of indoxyl-sulfate, quinolinic acid, kynurenine, N6-acetyl-lysine, dimethyl-guanosine, and adenosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises at least one metabolite selected from the group of neurotoxic compounds consisting of indoxyl-sulfate, kynurenine, and N6-acetyl-lysine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises at least one metabolite selected from the group of RBC enriched compounds consisting of ATP, S-adenosyl-methionine, NADP⁺, glutathione disulfide, pantothenate, and gluconate.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises at least one metabolite selected from the group of RBC enriched compounds consisting of ATP, NADP⁺, pantothenate, and gluconate.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is trimethyl-tryptophan.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-tryptophan and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, indoxyl-sulfate, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-tryptophan and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-tryptophan and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is S-methyl-ergothioneine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises S-methyl-ergothioneine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises S-methyl-ergothioneine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises S-methyl-ergothioneine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is betaine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises betaine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises betaine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises betaine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, and dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is indoxyl-sulfate.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises indoxyl-sulfate and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises indoxyl-sulfate and at least one metabolite selected from the group consisting of , kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises indoxyl-sulfate and at least one metabolite selected from the group consisting of kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is pantothenate.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises pantothenate and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺,keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises pantothenate and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises pantothenate and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is trimethyl-histidine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-histidine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-histidine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-histidine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises dimethyl-xanthine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises dimethyl-xanthine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises dimethyl-xanthine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, and betaine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is ATP.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises ATP and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises ATP and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises ATP and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is methionine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises methionine n and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises methionine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises methionine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is kynurenine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises kynurenine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises kynurenine and at least one metabolite selected from the group consisting of indoxyl-sulfate, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises kynurenine and at least one metabolite selected from the group consisting of indoxyl-sulfate, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is trimethyl-tyrosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-tyrosine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-tyrosine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-tyrosine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is trimethyl-phenylalanine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-phenylalanine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-phenylalanine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-phenylalanine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is NADP⁺.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises NADP⁺ and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises NADP⁺ and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises NADP⁺ and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is 2-hydroxybutyrate.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises 2-hydroxybutyrate and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises 2-hydroxybutyrate and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises 2-hydroxybutyrate and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is keto(iso)leucine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises keto(iso)leucine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises keto(iso)leucine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises keto(iso)leucine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is glycerophosphocholine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises glycerophosphocholine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises glycerophosphocholine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises glycerophosphocholine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is gluconate.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises gluconate and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises gluconate and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises gluconate and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is pseudouridine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises pseudouridine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises pseudouridine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises pseudouridine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is N6-acetyl-lysine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises N6-acetyl-lysine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises N6-acetyl-lysine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises N6-acetyl-lysine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is dimethyl-guanosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises dimethyl-guanosine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, and adenosine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises dimethyl-guanosine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for obtaining an index for the diagnosis of Alzheimer's disease (AD) by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises dimethyl-guanosine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

The method for obtaining an index for the diagnosis of Alzheimer's disease (AD) of the present invention comprises (i) a step of preparing a metabolomics sample, (ii) a step of measuring the content of the specific blood metabolites in the sample and (iii) a step of assisting in the diagnosis of AD in a subject.

(i) a step of preparing a sample
Metabolomic samples can be prepared as reported previously (NPL 4). All blood samples are drawn in a hospital laboratory to ensure rapid sample preparation. Briefly, venous blood samples for metabolomics analysis are taken into 5 mL heparinized tubes (Terumo). Immediately, 0.1~1.0 mL blood (4~60×10⁸ RBC) were quenched in 30~70% methanol (preferably 50~60%) of 5~10 times volume of the blood at -20°C~-80°C (preferably at -40°C~-50°C). This quick quenching step immediately after blood sampling ensured accurate measurement of many labile metabolites. The use of whole blood samples also allowed us to observe cellular metabolite levels that might otherwise have been affected by lengthy cell separation procedures. During Ficoll separation or leukodepletion by filtration, blood cells are exposed to non-physiological conditions for prolonged periods (NPL 4).

The remaining blood sample from each donor is centrifuged at 120 g for 15 min at room temperature to separate plasma and RBCs. After centrifugation, 0.1~1.0 mL each of separated plasma and RBCs (7-100x10⁸RBC), are quenched in 30~70% methanol (preferably 50~60%) of 5~10 times volume of the sample at -20°C~-80°C (preferably at -40°C~-50°C). Two internal standards (10 nmol of HEPES and PIPES) are added to each sample. After brief vortexing, samples are transferred to Amicon Ultra 10-kDa cut-off filters (Millipore, Billerica, MA, USA) to remove proteins and cellular debris. Thus, from each blood sample, three different subsamples, whole blood, RBCs, and plasma, are prepared. The white blood cell content (WBC) is less than 1% of the cellular volume in our preparations (NPL 4). Full metabolomics analysis of WBCs using a Ficoll gradient confirmed that WBCs should not affect our present metabolomics results regarding RBCs. After sample concentration by vacuum evaporation, each sample is re-suspended in 40 μL of 50% acetonitrile, and 1 μL is used for each injection into the LC-MS system.

(ii) a step of measuring
The content of blood metabolite in the sample of the subject is measured in this step. LC-MS data are preferably to be obtained using a Paradigm MS4 HPLC system (Michrom Bioresources, Auburn, CA, USA) coupled to an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), as previously described (Pluskal T, Nakamura T, Villar-Briones A, & Yanagida M (2010) Metabolic profiling of the fission yeast S. pombe: quantification of compounds under differenttemperatures and genetic perturbation. Mol Biosyst 6(1):182-198). Briefly, LC separation is performed on a ZIC-pHILIC column (Merck SeQuant, Umea, Sweden; 150 mm x 2.1 mm, 5 μm particle size). The HILIC column is quite useful for separating many hydrophilic blood metabolites, which are previously not assayed by others (NPL 4: Chaleckis R, et al. (2014)). Acetonitrile (A) and 10 mM ammonium carbonate buffer, pH 9.3 (B) are used as the mobile phase, with a gradient elution from 80-20% A in 30 min, at a flow rate of 100 μL mL-1. Peak areas of metabolites of interest are measured using MZmine 2 software (Pluskal T, Castillo S, Villar-Briones A, & Oresic M (2010) MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Bioinformatics 11:395). Detailed data analytical procedures and parameters have been described previously (Pluskal T, Nakamura T, Villar-Briones A, & Yanagida M (2010) Metabolic profiling of the fission yeast S. pombe: quantification of compounds under differenttemperatures and genetic perturbation. Mol Biosyst 6(1):182-198). Metabolomic datasets are deposited in the MetaboLights database (see data availability).

(iii) a step of assisting in the diagnosis of AD in a subject
The content of blood metabolite in the subject's sample measured in this step (ii) is compared to standard data, which is the average of the data for the blood metabolite in a healthy person, a healthy elderly person (HE) or a healthy young person (HY). When the content of blood metabolite in the subject's sample is apart from the standard, the subject could be judged to be AD or to be at high risk of AD. When the blood metabolites are indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine or dimethyl-guanosine, and when the content of one of the compounds in the subject’s blood sample is higher than standard, the subject could be diagnosed as having AD or is at high risk for AD. When the blood metabolites are ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine or tryptophan, and when the content of one of the compounds in the subject’s blood sample is lower than standard, the subject could be diagnosed as having AD or is at high risk for AD. However, the final diagnosis should be made by a doctor.

A method for assisting in the diagnosis of Alzheimer’s disease (AD)
The present invention is a method for assisting in the diagnosis of Alzheimer’s disease (AD) in a subject by measuring the amount of a specific blood metabolite in a subject. According to the present invention, by measuring the amount of the specific blood metabolite, it is possible to diagnose or assist in diagnosing whether a subject has AD or is at risk for AD. The section of "A method for obtaining an index for the diagnosis of Alzheimer's disease (AD)" can be referred for details of the invention of the method for assisting in the diagnosis of AD if needed.

Preferably, the blood metabolite comprises at least one metabolite selected from the group consisting of trimethyl-tryptophan, S-methyl-ergothioneine, betaine, indoxyl-sulfate, pantothenate, trimethyl-histidine, dimethyl-xanthine, ATP, methionine, kynurenine, trimethyl-tyrosine, trimethyl-phenylalanine, NADP⁺, 2-hydroxybutyrate, keto(iso)leucine, glycerophosphocholine, gluconate, pseudouridine, N6-acetyl-lysine, and dimethyl-guanosine.

More preferably, the blood metabolite comprises at least one metabolite selected from the group consisting of trimethyl-tryptophan, S-methyl-ergothioneine, betaine, indoxyl-sulfate, pantothenate, trimethyl-histidine, dimethyl-xanthine, ATP, methionine, and kynurenine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises at least one metabolite selected from the group of antioxidative aromatic compounds with trimethyl-ammonium consisting of ergothioneine, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, and trimethyl-tyrosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises at least one metabolite selected from the group of antioxidative aromatic compounds with trimethyl-ammonium consisting of S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, and trimethyl-tyrosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises at least one metabolite selected from the group of neurotoxic compounds consisting of indoxyl-sulfate, quinolinic acid, kynurenine, N6-acetyl-lysine, dimethyl-guanosine, and adenosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises at least one metabolite selected from the group of neurotoxic compounds consisting of indoxyl-sulfate, kynurenine, and N6-acetyl-lysine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises at least one metabolite selected from the group of RBC enriched compounds consisting of ATP, S-adenosyl-methionine, NADP⁺, glutathione disulfide, pantothenate, and gluconate.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises at least one metabolite selected from the group of RBC enriched compounds consisting of ATP, NADP⁺, pantothenate, and gluconate.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is trimethyl-tryptophan.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-tryptophan and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, indoxyl-sulfate, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-tryptophan and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-tryptophan and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, pantothenate, betaine, dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is S-methyl-ergothioneine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises S-methyl-ergothioneine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises S-methyl-ergothioneine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises S-methyl-ergothioneine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is betaine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises betaine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises betaine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises betaine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, and dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is indoxyl-sulfate.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises indoxyl-sulfate and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises indoxyl-sulfate and at least one metabolite selected from the group consisting of , kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises indoxyl-sulfate and at least one metabolite selected from the group consisting of kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is pantothenate.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises pantothenate and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺,keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises pantothenate and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises pantothenate and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is trimethyl-histidine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-histidine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-histidine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-histidine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises dimethyl-xanthine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises dimethyl-xanthine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises dimethyl-xanthine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, and betaine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is ATP.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises ATP and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises ATP and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises ATP and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is methionine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises methionine n and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises methionine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises methionine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is kynurenine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises kynurenine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises kynurenine and at least one metabolite selected from the group consisting of indoxyl-sulfate, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises kynurenine and at least one metabolite selected from the group consisting of indoxyl-sulfate, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is trimethyl-tyrosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-tyrosine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-tyrosine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-tyrosine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is trimethyl-phenylalanine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-phenylalanine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-phenylalanine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises trimethyl-phenylalanine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is NADP⁺.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises NADP⁺ and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises NADP⁺ and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises NADP⁺ and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is 2-hydroxybutyrate.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises 2-hydroxybutyrate and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises 2-hydroxybutyrate and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises 2-hydroxybutyrate and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is keto(iso)leucine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises keto(iso)leucine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises keto(iso)leucine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises keto(iso)leucine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is glycerophosphocholine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises glycerophosphocholine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises glycerophosphocholine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises glycerophosphocholine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is gluconate.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises gluconate and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises gluconate and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises gluconate and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is pseudouridine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises pseudouridine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises pseudouridine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises pseudouridine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is N6-acetyl-lysine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises N6-acetyl-lysine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, pseudouridine, adenosine and dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises N6-acetyl-lysine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises N6-acetyl-lysine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite is dimethyl-guanosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises dimethyl-guanosine and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, and adenosine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises dimethyl-guanosine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, dimethyl-xanthine, quinolinic acid, adenosine, trimethyl-histidine, trimethyl-tyrosine, trimethyl-phenylalanine, S-adenosyl-methionine, glutathione disulfide, keto(iso)leucine, and caffeine.

One embodiment of the present invention is a method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject, wherein the specific blood metabolite comprises dimethyl-guanosine and at least one metabolite selected from the group consisting of indoxyl-sulfate, kynurenine, ergothioneine, S-methyl-ergothioneine, trimethyl-tryptophan, pantothenate, betaine, and dimethyl-xanthine

The method for assisting in the diagnosis of AD in a subject by measuring the amount of a specific blood metabolite in a subject of the present invention comprises (i) a step of preparing a metabolomics sample, (ii) a step of measuring the content of the specific blood metabolites in the sample and (iii) a step of assisting in the diagnosis of AD in a subject. The section of "A method for obtaining an index for the diagnosis of Alzheimer's disease (AD)" can be referred for details of each step of the invention of the method for assisting in the diagnosis of AD.

Apparatus
The present invention provides an apparatus for obtaining an index for the diagnosis of Alzheimer's disease (AD). The present invention also provides an apparatus for assisting in the diagnosis of AD in a subject. The apparatus uses the method of the present invention above.

The apparatus for obtaining an index for the diagnosis of Alzheimer's disease (AD) of the present invention comprises means for input and means for assisting in the diagnosis of AD in a subject, comprising measuring the amount of a specific blood metabolite in the subject, wherein data of blood metabolites of the subject are input to the means for input, and the diagnosis of AD is performed by comparing standard. Said method section can be referred for details of the method of the present invention used by the apparatus.

System
The present invention provides a system for obtaining an index for the diagnosis of Alzheimer's disease (AD). The present invention also provides a system for assisting in the diagnosis of AD in a subject. The system uses the method of the present invention above and/or the apparatus of the present invention. Said method section and the apparatus section can be referred for details of the system of the present invention.

Methods
The present invention provides a method of evaluating substances which could be used in treating AD comprising the step of measuring a blood metabolite, wherein the blood metabolite comprises at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine. The substances found by this evaluation method can be widely used as foods, drinks, supplements, pharmaceuticals for improving the condition of AD. The section of "A method for obtaining an index for the diagnosis of Alzheimer's disease (AD)" can be referred for details of the step of measuring a blood metabolite.

Kit
The present invention provides a kit for obtaining an index for the diagnosis of Alzheimer's disease (AD) or assisting in the diagnosis of AD by using the methods of the present invention, comprising blood collection tubes and blood metabolite compounds as detection standard. The kit of the present invention may comprise any constituent elements besides the blood collection tube and the like. The blood metabolite compounds as detection standard can be selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, indoxyl-sulfate, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.

1. Collection of blood samples from Alzheimer’s disease (AD) subjects
To identify AD-related blood metabolites, quantitative comparison was conducted for the samples of AD patients, healthy elder (HE) and healthy young (HY) subjects. Blood samples of AD patients (age, 75 ~ 88yr) diagnosed as AD and hospitalized at the National Hospital Organization Ryukyu Hospital, Kin, Okinawa were obtained for each patient after informed consent was obtained. The same number of HE (67 ~ 80yr) and HY (28 ~ 34yr) volunteers from Onna Clinic, Onna-son in Okinawa were recruited (Table 1).

Total 24 subjects comprised of 8 AD patients, 8 healthy elderly (HE) and 8 healthy young (HY) subjects participated the present study. Table 1 shows their age, gender, BMI, and 2 types of dementia test data. The mini mental health examination (MMSE) and Hasegawa’s dementia scale-revised (HDS-R) were employed to assess cognitive ability of AD subjects. In Figure 1, age and BMI are schematically shown. Ages of AD subjects ranged from 75 to 88 yr and their BMIs varied from 14.8 to 24.5. The 8 HE subjects had an age range from 67-80 with a BMI range from 17-25. The 8 HY subjects were between 28 to 34 yr with BMI 21 to 24.

2. Thirty-three metabolites notably differed between AD and HE
In all whole blood samples collected, 124 metabolites could be identified and quantified by non-targeted, comprehensive LC-MS methods (Table 2-1 to 2-3).

They consisted of 14 subgroups; 1. Nine nucleotides, 2. Twelve nucleosides, nucleobases, and derivatives, 3. Five vitamins and coenzymes, 4. Four nucleotide-sugar derivatives, 5. Eleven sugar phosphates, 6. Four sugar derivatives, 7. Seven choline and ethanolamine derivatives, 8. Ten organic acids, 9. Two anti-oxidants, 10. Seventeen standard amino acids, 11. Fourteen methylated amino acids, 12. Four acetylated amino acids, 13. Sixteen other amino acids, 14. Nine carnitines. Fifty-one compounds enriched in RBCs are underlined in the table. The majorities of 5 subgroups (nucleotides, vitamins and coenzymes, nucleotides-sugar derivatives, sugar phosphates, and anti-oxidants) are RBC-enriched (Chaleckis et al., 2016). Amounts of metabolites were obtained by measuring peak areas for each compound (ion number; denoted by H, M and L, for high (10⁸), medium (10⁷) and low (10⁶) levels, respectively). Total 33 metabolites that significantly differed statistically between AD and HE (indicated with asterisks) are listed with p-values (0.00016< p <0.05), and shown as AD markers in Table3. To estimate of false discovery rate, q-values were calculated. Q-values were mostly consistent with p-values of 33 metabolites. Five compounds, ATP, Glutathione disulfide, glutamine, phenylalanine, and betaine are highly abundant (ranked H). Five compounds, glycerophosphocholine, ergothioneine, methionine, tryptophan, and tyrosine are subsequently abundant (H-M). Abundances of following 3 compounds are broadly distributed (H-L); caffeine, dimethyl-xanthine, and trimethyl-tryptophan. Remaining 20 compounds are medium to low abundance (M-L, M, L). Twelve compounds underlined are RBC-enriched. Nine characteristic AD-compounds with circle contained trimethylated ammonium; six of them are methylated amino acids. Others with circle are anti-oxidant (ergothioneine), choline (glycerophosphocholine) and carnitine (dodecanoyl-carnitine) (Table 3).

Mann Whitney U-test was performed to obtain p-values. Thus all 33 AD markers showed p-values less than 0.05. Resulting peak ratios calculated using the median of peak abundance in AD and HE are shown in the 4th column of Table 3 (ratio). Seven compounds that increased in AD showed the values more than 1.0. Otherwise ratios are less than 1.0. Four compounds greatly declined are dimethyl-xanthine (0.04), trimethyl-tyrosine (0.08), caffeine (0.09), trimethyl-tryptophan (0.10). These lessened representative compounds are aromatics. Their distributions are broad from H to L in healthy subjects.

Seven compounds increased in the AD as shown in Table 3 (AD/HE ratio>1.0). Examples are indoxyl-sulfate (1.93), quinolinic acid (1.79), dimethyl-guanosine (1.45), pseudouridine (1.19), and kynurenine (1.12). These metabolites were reported to be toxic (Vanholder et al., 2003) (see below).

3. Dot plot profiles of 26 blood compounds decreased in AD subjects
Figure 2-1 to 2-13 shows dot plot profiles of 26 compounds’ abundances in AD, HE and HY. They all declined in AD patients compare to HE (p<0.05). Ergothioneine (anti-oxidant, a thiourea derivative of trimethyl histidine), glutathione disulfide (redox compound) (Martinez de Toda et al., 2019), betaine (a zwitter ion containing trimethyl-glycine) and ATP (energy carrier) were found to be most abundant metabolites that decreased in AD. Two other ergothioneine-related, but less abundant compounds, S-methyl-ergothioneine and trimethyl-histidine (hercynine), also declined in AD.

Trimethyl-tryptophan (hypaphorine), trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine (Cristofano et al., 2016), and trimethyl-tyrosine, all of which contain trimethylammonium ions also declined, strongly suggesting that these trimethyl-ammonium compounds were characteristically declined possibly due to their instability or declined synthesis or import declined in human body in AD patients. Caffeine and its derivative, dimethyl-xanthine, uridine (a pyrimidine nucleoside) (Olde Rikkert et al., 2014), S-adenosyl-methionine (methyl donor) (Guiraud et al., 2017), NADP⁺ (oxidoreductive coenzyme), pantothenate (vitamin B5), keto(iso)leucine (keto-acid), 2-hydroxybutyrate (lipid-degradation product) and gluconate (organic acid bound to Zn), glutamine (Gonzalez-Dominguez et al., 2014, 2015), phenylalanine (Gonzalez-Dominguez et al., 2015; Mapstone et al., 2014), tyrosine (Gonzalez-Dominguez et al., 2015), histidine (Gonzalez-Dominguez et al., 2014, 2015), methionine, tryptophan (regular amino acids) (Gonzalez-Dominguez et al., 2015; Gulaj et al., 2010) all diminished in AD subjects. Among them, the degree of reduction for caffeine and dimethyl-xanthine in AD was quite significant (ratio, 0.04 ~ 0.09). Trimethyl-tryptophan (0.10) and trimethyl-tyrosine (0.08) were also strikingly declined.

4. Nine trimethylated ammonium compounds were declined in AD patients
In Figure 3, nine trimethylated compounds that decreased in AD are schematically shown. Trimethylated ammonium ion moiety is marked by orange color. Three of them (betaine, glycerophosphocholine, and dodecanoyl carnitine) are synthesized in human body, whereas other six trimethylated compounds containing aromatic portion are dietary derived compounds. Most strikingly six of these nine compounds are abundant (H, H-M or H-L) enriched in plasma and RBCs. Hence the sharp decline of these amphipathic compounds (possessing both hydrophilic and lipophilic properties and forming basis of lipid polymorphism) in AD brain may affect considerably the physicochemical properties of neuronal systems in AD patients.

5. Seven metabolites increased in AD
Interestingly, 7 metabolites, comprising 3 nucleosides and 4 amino acid derivatives, increased in AD (Figure 2-14 to 2-17). None was highly abundant and none was enriched in RBCs, so that their increase in AD occurred in plasma. Indoxyl-sulfate, kynurenine and quinolinic acid (Gulaj et al., 2010) are involved in tryptophan metabolism and possibly acting as excitatory toxins in brain (Adesso et al., 2017; Stone et al., 2003), while N6-acetyl-lysine is implicated in histone and non-histone protein modification (Iwabata et al., 2005). Pseudouridine, adenosine (Gonzalez-Dominguez et al., 2015), and dimethyl-guanosine are degradation products of RNAs present in urine and thought to be oxidized (Lee et al., 2007; Sander et al., 1986). Increases of these metabolites in AD are of interest, as some are reportedly toxic in the central nervous system (CNS) and may lead to impairment in the brain (Moroni, 1999; Rahman, 2009; Ruddick et al., 2006). Their increase in brain might be disadvantageous and detrimental.

6. PCA separates AD patients from healthy elderly (HE)
To distinguish between AD and HE subjects, we then applied principal component analysis (PCA). We calculated principal component (PC1, PC2) values using the abundance data of the 33 AD-related metabolites in 16 subjects. Figure 4-1 shows that two classes of subjects AD (red triangles) and non-AD (HE, green triangles) were clearly separated between the groups of AD and non-AD subjects. Thus PCA was capable to separate AD from non-AD if 33 AD compounds were used for calculation. We then reduced the number of compounds examined and found that levels of only six metabolites (dimethyl-guanosine, pseudouridine, NADP⁺, trimethyl-histidine, ergothioneine, S-methyl-ergothioneine) were able to separate AD from non-AD subjects almost perfectly (Figure 4-2). Thus the PCA method was useful for separating AD patients from non-AD subjects using even small number of AD components.

7. Correlation analysis revealed five metabolite groups
To gain insight into how these 33 AD metabolites are related each other, we searched for relationships among AD metabolites using Pearson’s correlation function r. Significant positive and negative correlations (0.89> r >0.50 or -0.68 < r <-0.50) were found to be present among 33 metabolites. They are highlighted in Figure 5 (positive ; grey without “X”, negative; grey with “X”). It was noticed that five of seven increased AD markers were correlated (0.89~0.57) among quinolinic acid, dimethyl-guanosine, pseudouridine, indoxyl-sulfate, and kynurenine. This striking finding of high correlation validated at least partly the classification of subgroup A. Further, these compounds are negatively correlated (-0.66 ~ -0.50) with many other lower subgroup B, C, and D compounds, consistent with presumed opposing tendency of these compounds to group A.

Interestingly, negative correlations exist between group A and group B compounds that contained trimethylated ammonium metabolites, and between group A and group C compounds comprised of RBC-enriched metabolites containing oxidoreductive (NADP⁺, glutathione disulfide), methyl-donor (S-adenosyl-methionine), binding to iron and zinc (pantothenate, gluconate) and energy carrier (ATP). Group B and C metabolites are correlated within the groups, again validating the classification of B and C. Group B metabolites are structurally related as they commonly contain trimethylated-ammonium group indicating that they are anti-oxidative. Group C are not structurally related but three of them (NADP⁺, glutathione disulfide and ATP) implicated in redox and energy metabolism are highly correlated and enriched in RBC. Gluconate also enriched in RBCs is known to bind to zinc and is correlated (0.56) to histidine that is known to bind to zinc. Fourteen group D metabolites are enriched in plasma and, as shown in Table 3, are internally correlated such as among regular amino acids, betaine, nucleoside, keto(iso)leucine. For example, correlation between tryptophan and tyrosine is 0.72. Remaining compounds (2-hydroxybutyrate, dodecanoyl-carnitine) are hardly correlated. Finally, two highly related metabolites group E (caffeine, dimethyl-xanthine; r, 0.76) were notified. There are reports on caffeine showing that they are beneficial to relieve AD (see Discussion). Thus 33 AD metabolites were grouped into 5 A-E subgroups. They are mostly positively correlated internally, but group A interacts negatively with B-E in different degree.

8. Heatmap presentation of AD subjects in comparison with HE
Using abundance data of 33 AD-compounds in 8 AD patients and 8 HE subjects, a heatmap was constructed (Figure 6). In AD patients (#1~8), 7 compounds (from quinolinic acid to adenosine) increased, whereas remaining 26 compounds decreased (see the figure caption). Indeed the majority of AD subjects revealed more than average map units (>50) for the 7 increased compounds, whilst in HE subjects, the majority of map units for increased seven compounds showed the levels below the average (<50), providing evidence for the decrease of these compounds. For subject #6 (an AD patient), the abundance of N6-acetyl-lysine was close to the average (47.9). Otherwise other 6 metabolites are more than average (>50, 52~80) showing the grey color without “X” cells. It is striking the seven compounds nearly uniformly increased in the AD patients, whereas in HE subjects, the levels were mostly below 50, representing the grey with “X” decline from the average. Thus these seven markers indeed increased in AD patients, but decreased in healthy subjects. These seven compounds are appropriate candidates for the increase markers of AD.

On the other hand, the abundances of 26 metabolites from S-methyl-ergothioneine at the top to dimethyl-xanthine at the bottom were largely below the average (<50, showing grey with “X” cells) in AD patients. In sharp contrast, the majorities in HE subjects showed their levels more than average (>50, grey without “X”). Glutathione disulfide, NADP⁺ and ergothioneine showed the sharpest separation between AD and HE, suggesting that the decline of oxido-reductive and anti-oxidative compounds may be the principal decline in AD subjects among 26 AD blood compounds.

(Materials, Methods and Procedures)
Participants
Healthy 8 young and 8 elderly volunteers, and 8 AD patients who hospitalized at the National Hospital Organization Ryukyu Hospital participated as subjects in this study (information summarized in Table 1).

Ethics statement
Witten, informed consent was obtained from all participants in accordance with the Declaration of Helsinki. All experiments were performed in compliance with relevant Japanese laws and institutional guidelines. All protocols were approved by the Ethics Committee on Human Research of Ryukyu Hospital and by the Human Subjects Research Review Committee of the Okinawa Institute of Science and Technology Graduate University (OIST).

Chemicals and reagents
Standards for metabolite identification were purchased from commercial sources as described previously (Chaleckis et al., 2014; Chaleckis et al., 2016; Teruya et al., 2019). LC-MS grade acetonitrile, methanol, and ultrapure water were obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

Blood sample preparation
Metabolomic samples were prepared as described previously. Briefly, venous blood samples were taken in to heparinized tubes before breakfast in the morning. Subjects were asked to ensure at least 8 hr of fasting prior to sampling. During fasting, they took water freely. Immediately, 0.2 mL of blood were quenched in 1.8 mL of 55% methanol at -40°C. Ten nmol each of HEPES and PIPES were added to each sample to serve as standards, After brief vortexing, samples were transferred to Amicon Ultra 10-kDa cut-off filters (Millipore, Billerica, MA, USA) to remove proteins and cellular debris. After sample concentration by vacuum evaporation, each sample was re-suspended in 40 μL of 50% acetonitrile, and 1 μL was used for each injection into the LC-MS system, as described.

LC-MS analysis
Non-targeted LC-MS conditions were as described previously. Briefly, LC-MS data were obtained using an Ultimate 3000 DGP-3600RS HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled to an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). LC separation was performed on a ZIC-pHILIC column (Merck SeQuant, Umea, Sweden; 150 mm × 2.1 mm, 5 μm particle size). Acetonitrile (A) and 10 mM ammonium carbonate buffer, pH 9.3 (B) were used as the mobile phase, with a linear gradient elution from 80-20% A over 30 min, at a flow rate of 100 μL/mL. The mass spectrometer was operated in full-scan mode with a 100-1000 m/z scan rate and automatic data-dependent MS/MS fragmentation scans.

LC-MS data processing and peak characteristics
Peak areas of metabolites of interest were measured using MZmine 2 software (Pluskal et al., 2010). Data analytical procedures and parameters have been described previously. We analyzed 124 blood metabolites that were confirmed using standards or MS/MS analysis. According to their peak areas, metabolite abundances were classified into 3 groups (H, M, and L). H denotes compounds with high peak areas (>10⁸ AU), M with medium peak areas (10⁷~10⁸ AU) and L with low peak areas (<10⁷ AU) (Table 2-1 to 2-5).

Statistical analysis
Peak data processed by MZmine 2 were exported into spreadsheet format and analyzed with R statistical software (http://www.r-project.org). Statistical analysis was performed using the Mann Whitney U-test. Statistical significance was established at p<0.05. Q-values were calculated using the Benjamini-Hochberg method. The score plot of principal component (PC) was generated by SIMCA-P+ software (Umetrics Inc., Umea, Sweden). Heatmap represents standardized abundance data for each metabolite. T-scores were calculated from the following formula: T-score = [(sample peak area - average of population peak area) × 10/standard deviation of population peak area] + 50. Therefore, mean and standard deviation are 50 and 10, respectively.

Data and materials availability
Raw LC-MS data in mzML format are accessible via the MetaboLights repository (http://www.ebi.ac.uk/metabolights). Data for the 24 subjects are available under accession number MTBLS2109.

(Discussion)
In this study, we showed that 26 metabolites, which declined in AD blood, contained oxidoreductive, anti-oxidative, energy supplying, and trimethyl-ammonium compounds. Specifically, glutathione disulfide, NADP⁺, ATP, trimethyl-tryptophan, trimethyl-tyrosine, trimethyl-phenylalanine, ergothioneine and S-methyl-ergothioneine caused the sharpest separation between AD and HE subjects in their abundance. In contrast, we found the increased 7 AD markers (quinolinic acid, kynurenine, adenosine, dimethyl-guanosine, indoxyl-sulfate and pseudouridine and N6-acetyl-lysine) which likely act as inhibitors or poisons to brain functions. Thus comprehensive metabolomics and Pearson’s correlation analysis revealed two types of metabolites (subgroup A and B-E) that showed opposing behavior in abundance.

These total 33 AD marker metabolites were identified in whole blood of AD patients in comparison with HE. We employed statistical analyses (p- and q-values) both of which confirmed the statistical results. Correlation analysis allowed us to categorize them into five (A-E) subgroups (Figure 5). Seven group A compounds increased in AD patients, whereas 26 compounds (B-E sub-groups) decreased. In the literature, 11 of them had been already reported to be implicated in AD so that 22 are novel (indicated in Table 3). Seven A compounds contained oxidized compounds related to tryptophan degradation and nucleosides metabolism. Five of them are highly correlated. Judging from the literature, they may act as inhibitors and/or poisons or wastes (Mutsaers et al., 2013; Vanholder et al., 2003). Kynurenine, quinolinic acid (Guillemin and Brew, 2002) and indoxyl-sulfate may target brain and worsen AD (Adesso et al., 2017). N6-acetyl-lysine may be related to accumulation of acetylated tau and a-tubulin in AD brain (Cohen et al., 2013; Zhang et al., 2015).

Six group B compounds contained trimethyl ammonium moiety including ergothioneine, presumably acting as anti-oxidants, so that group B may act against group A, concomitantly against AD. Hence declining of group B compounds might also cause the progression of AD (Figure 7). Similar result was obtained for frailty patients having mild cognitive impairment (Kameda et al., 2020). The decline of ergothioneine, S-methyl-ergothioneine, trimethyl-histidine, tryptophan and methionine was observed in frailty that is impaired in cognitive ability. Six group C are enriched in RBCs, consisting of glutathione, NADP⁺, pantothenate, S-adenosyl-methionine, energy carrier and metal (Zinc) carrier. Their actions may directly or indirectly resist against AD. Twelve group D compounds containing amino acids, nucleosides, choline, and carnitine are plasma enriched compounds and may underpin the actions of other metabolites for supply and degradation. The last two E compounds are caffeine and dimethyl-xanthine (see below). Thus we consider these B-E metabolites are, if not all, involved in restraining AD development (Figure 7).

Caffeine, an anti-oxidant purine, and its derivative, dimethyl-xanthine (highly 0.76 correlated) are reduced in AD subjects. Their relationship to AD has been investigated, as caffeine may be a possible protectant against cognitive decline (Kolahdouzan and Hamadeh, 2017; Sc and Muralidhara, 2016), because of its anti-oxidative purine activity. We showed here that adenosine belonged to group A and increased its level 1.5-fold in AD. Caffeine is known to be antagonist of adenosine (Ribeiro and Sebastiao, 2010). Hence it may be explained why caffeine was beneficial for AD patients (Flaten et al., 2014; Rahman, 2009), as caffeine opposes adenosine.

In AD subjects, mitochondrial dysfunction has been reported, so ATP levels may diminish (Butterfield and Halliwell, 2019; Cadonic et al., 2016). Indeed the level of ATP in this study was reduced (0.83) in AD subjects, particularly clear in the heatmap result (Figure 6). ATP was clearly diminished in AD, whereas the level was near average or above the average in HE subjects (Figure 6). In correlation analysis, ATP concentrations were positively correlated (r>0.50) with 10 compounds in subgroups B, C, and D (Figure 5) so that the level of ATP may affect or be affected by concentrations of many metabolites including oxidoreductive compounds, glutathione, betaine, ergothioneine, S-methyl-ergothioneine and amino acids such as glutamine, tryptophan. Thus it is quite possible that ATP might assist anti-oxidation in brain via many metabolites. NADP⁺ and glutathione disulfide may be synergistic to maintain the level of ATP and ergothioneine. Hence, oxido-reductive NADP⁺, anti-oxidative glutathione and presumably neuroprotective trimethylated ammonium compounds may all together function in an overlapped fashion to sustain brain mitochondrial ATP production level against AD. Note that glycerophosphocholine and dodecanoyl-carnitine belonged to group D might also positively affect mitochondrial function.

Total 9 compounds possessing trimethylated ammonium ion (illustrated in Figure 3) are amphipathic compounds (possessing both hydrophilic and lipophilic properties) and forming basis of lipid polymorphism and all of them showed sharp decline in their abundance in AD subjects. The cause of decline might be due to ROS (reactive oxygen species) effect in AD patients’ brain. These compounds may have some similar role, such as amphipathic compounds forming the highly ordered assembled structure. In addition, these compounds are abundant (the level H, H-M, and H-L). They might act as major neuroprotectants or antioxidants in brain, and their levels are sensitive to both anti-oxidants and ROS. In addition, membrane defects have been observed in AD patients’ brain that degrade glycerophosphocholine (Nitsch et al., 1992).

An advantage of metabolomic analysis over proteomic and genomic studies exists in the relationship to search drugs. As metabolites exist in human body, they may be employed for the study on drug therapy. In the present study, 33 metabolites are obvious targets of future study. Their majority has not been studied at all for their potential for clinical purpose of AD.

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Transl Neurodegener

4, 9.

The present invention based on these findings provides a novel method capable of simple and accurate diagnosis of Alzheimer's disease (AD). The present invention also provides a novel method for obtaining an index for the diagnosis of Alzheimer’s disease (AD). The present invention also provides a novel method for assisting in the diagnosis of AD in a subject, comprising measuring the amount of a specific blood metabolite in the subject.

Claims

A method for obtaining an index for the diagnosis of Alzheimer’s disease (AD) by measuring at least one blood metabolite selected from the group consisting of trimethyl-tryptophan, S-methyl-ergothioneine, betaine, indoxyl-sulfate, pantothenate, trimethyl-histidine, dimethyl-xanthine, ATP, methionine, kynurenine, trimethyl-tyrosine, trimethyl-phenylalanine, NADP⁺, 2-hydroxybutyrate, keto(iso)leucine, glycerophosphocholine, gluconate, pseudouridine, N6-acetyl-lysine, and dimethyl-guanosine.
The method for obtaining an index for the diagnosis of Alzheimer’s disease (AD) according to claim 1, wherein the blood metabolite is trimethyl-tryptophan.
The method for obtaining an index for the diagnosis of Alzheimer’s disease (AD) according to claim 1, wherein the blood metabolite is trimethyl-tryptophan and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, indoxyl-sulfate, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.
The method for obtaining an index for the diagnosis of Alzheimer’s disease (AD) according to claim 1, wherein the blood metabolite is selected from the group of antioxidative aromatic compounds with trimethyl-ammonium consisting of S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, and trimethyl-tyrosine.
The method for obtaining an index for the diagnosis of Alzheimer’s disease (AD) according to claim 1, wherein the blood metabolite is selected from the group of neurotoxic compounds consisting of indoxyl-sulfate, kynurenine, and N6-acetyl-lysine.
The method for obtaining an index for the diagnosis of Alzheimer’s disease (AD) according to claim 1, wherein the blood metabolite is selected from the group of RBC enriched compounds consisting of ATP, NADP⁺, pantothenate, and gluconate.
A method for assisting in the diagnosis of AD in a subject by measuring at least one blood metabolite selected from the group consisting of trimethyl-tryptophan, S-methyl-ergothioneine, betaine, indoxyl-sulfate, pantothenate, trimethyl-histidine, dimethyl-xanthine, ATP, methionine, kynurenine, trimethyl-tyrosine, trimethyl-phenylalanine, NADP⁺, 2-hydroxybutyrate, keto(iso)leucine, glycerophosphocholine, gluconate, pseudouridine, N6-acetyl-lysine, and dimethyl-guanosine.
The method for assisting in the diagnosis of AD in a subject according to claim 7, wherein the blood metabolite is trimethyl-tryptophan.
The method for assisting in the diagnosis of AD in a subject according to claim 7, wherein the blood metabolite is trimethyl-tryptophan and at least one metabolite selected from the group consisting of ergothioneine, glutathione disulfide, betaine, ATP, S-methyl-ergothioneine, trimethyl-histidine, indoxyl-sulfate, trimethyl-phenylalanine, glycerophosphocholine, dodecanoyl-carnitine, trimethyl-tyrosine, caffeine, dimethyl-xanthine, uridine, S-adenosyl-methionine, NADP⁺, pantothenate, keto(iso)leucine, 2-hydroxybutyrate, gluconate, glutamine, phenylalanine, tyrosine, histidine, methionine, tryptophan, kynurenine, quinolinic acid, N6-acetyl-lysine, pseudouridine, adenosine and dimethyl-guanosine.
The method for assisting in the diagnosis of AD in a subject according to claim 7, wherein the blood metabolite is selected from the group of antioxidative aromatic compounds with trimethyl-ammonium consisting of S-methyl-ergothioneine, trimethyl-histidine, trimethyl-tryptophan, trimethyl-phenylalanine, and trimethyl-tyrosine.
The method for assisting in the diagnosis of AD in a subject according to claim 7, wherein the blood metabolite is selected from the group of neurotoxic compounds consisting of indoxyl-sulfate, kynurenine, and N6-acetyl-lysine.
The method for assisting in the diagnosis of AD in a subject according to claim 7, wherein the blood metabolite is selected from the group of RBC enriched compounds consisting of ATP, NADP⁺, pantothenate, and gluconate.
An apparatus for obtaining an index for the diagnosis of Alzheimer’s disease (AD) in which an index for the diagnosis of Alzheimer’s disease (AD) is obtained by the method according to any one of claims 1 to 6.
A system for obtaining an index for the diagnosis of Alzheimer’s disease (AD) in which AD is evaluated by the method according to any one of claims 1 to 6 or the apparatus according to claim 13.
An apparatus for assisting in the diagnosis of AD in a subject in which the diagnosis of AD is assisted by the method according to any one of claims 7 to 12.
A system for assisting in the diagnosis of AD in a subject in which the diagnosis of AD is assisted by the method according to any one of claims 7 to 12 or the apparatus according to claim 15.
A method of evaluating substances which improve Alzheimer’s disease (AD) comprising the step of measuring a blood metabolite such as trimethyl-tryptophan, S-methyl-ergothioneine, betaine, indoxyl-sulfate, pantothenate, trimethyl-histidine, dimethyl-xanthine, ATP, methionine, kynurenine, trimethyl-tyrosine, trimethyl-phenylalanine, NADP⁺, 2-hydroxybutyrate, keto(iso)leucine, glycerophosphocholine, gluconate, pseudouridine, N6-acetyl-lysine, or dimethyl-guanosine as AD markers.
A kit for evaluation of Alzheimer’s disease (AD) in which AD is evaluated by the method according to any one of claims 1 to 12 comprising blood collection tubes and blood metabolite compounds as detection standard.