WO2019208588A1 - Novel trimethyl ammonium metabolites in human blood cells that increase by aging - Google Patents

Abstract

It is an object of the present invention to provide a novel method capable of simply and accurately determining the extent of aging. The present invention is related to a method for determining the extent of aging in which a blood metabolite is used as an indicator. Preferably, at least one selected from the group consisting of whole blood, Red blood cells and plasma from a subject is used as a sample, and a blood metabolite in the sample is used as an indicator.

Description

NOVEL TRIMETHYL AMMONIUM METABOLITES IN HUMAN BLOOD CELLS THAT INCREASE BY AGING

The present invention relates to a method for determining the extent of aging, an apparatus for determining the extent of aging, a system for determining the extent of aging, a kit for determining the extent of aging and a method of evaluating substances which affect the extent of aging.

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.

While blood consists of noncellular (plasma or serum) and cellular components, most human blood metabolomics studies have focused on plasma or serum, for which large biobanks (curated collections of samples of plasma, urine, etc.) are now available (NPL 4-8). These studies are useful to understand disease mechanisms and to identify diagnostic markers for diseases, such as diabetes (NPL 9). Some genome-wide studies have also employed metabolomics (reviewed in Kastenmueller et al 2015 (NPL 10)). In contrast, few comprehensive metabolomics reports exist regarding red blood cells (RBCs) [e.g. Nishino et al 2009 (NPL 11)], although RBCs comprise nearly half the blood volume. This is partly due to technical difficulties in stabilizing labile cellular metabolites (NPL 12).

Recently we reported that 14 blood metabolites that strikingly differ in abundance between young (~30 yr) and elderly (70~95 yr) groups using non-targeted, comprehensive LC-MS (NPL 13). Six of them are RBC-enriched, suggesting that RBC
metabolomics is highly valuable for human aging research. It can be partly explained that antioxidant production is decreased and inefficiency of urea metabolism is increased among the elderly. These blood metabolites may be important to molecularly characterize the degree of human aging as they may reflect certain physiological states of human aging that is influenced in blood by genetic, epigenetic, and life-style factors. It is desirable that the aging marker shows a more pronounced quantitative change depending on the degree of aging.

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. Dunn WB, et al. (2015) Molecular phenotyping of a UK population: defining the human serum metabolome. Metabolomics 11:9-26. Guertin KA, et al. (2014) Metabolomics in nutritional epidemiology: identifying metabolites associated with diet and quantifying their potential to uncover diet-disease relations in populations. Am J Clin Nutr 100(1):208-217. Lawton KA, et al. (2008) Analysis of the adult human plasma metabolome. Pharmacogenomics 9(4):383-397. Psychogios N, et al. (2011) The human serum metabolome. PLoS One 6(2):e16957. Yu Z, et al. (2012) Human serum metabolic profiles are age dependent. Aging Cell 11(6):960-967. Suhre K (2014) Metabolic profiling in diabetes. J Endocrinol 221(3):R75-85. Kastenmuller G, Raffler J, Gieger C, & Suhre K (2015) Genetics of human metabolism: an update. Hum Mol Genet 24(R1):R93-R101. Nishino T, et al. (2009) In silico modeling and metabolome analysis of long-stored erythrocytes to improve blood storage methods. J Biotechnol 144(3):212-223. Gil A, et al. (2015) Stability of energy metabolites-An often overlooked issue in metabolomics studies: A review. Electrophoresis 36(18):2156-2169. Chaleckis R, et al. (2016) Individual variability in human blood metabolites identifies age-related differences. Proc Natl Acad Sci U S A 113(16):4252-4259. Pluskal T, Nakamura T, Villar-Briones A, & Yanagida M (2010) Metabolic profiling of the fission yeast S. pombe: quantification of compounds under different temperatures and genetic perturbation. Mol Biosyst 6(1):182-198. 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.

It is an object of the present invention to provide novel metabolites strongly associating with healthy aging for determining the extent of biological aging.

We employed analysis of metabolites by non-targeted metabolomics that provides comprehensive information of metabolites abundance. In the targeted analysis, metabolites for analysis are pre-determined so that abundance changes of untargeted metabolites may be overlooked. Although the non-targeted analysis is far more time consuming than targeted LC-MS, it is worth trying for the discovery of known or unknown compounds’ abundance change.

In the present invention, we found novel nine blood metabolites for determining the extent of aging in which a blood metabolite is used as an indicator.

The present inventions are as follows.
[1] A method for determining the extent of aging in which a blood metabolite is used as an indicator.
[2] The method for determining the extent of aging according to [1], wherein whole blood or Red blood cells from a subject are used as a sample, and a blood metabolite in the sample is used as an indicator.
[3] The method for determining the extent of aging according to [2], wherein the sample is treated with cold organic solvent immediately after bleeding.
[4] The method for determining the extent of aging according to any one of [1] to [3], wherein the blood metabolite comprises at least one metabolite selected from the group consisting of compound B, compound C, compound D, compound F, compound G, compound H, compound I, compound J, and compound L shown in Table 1.

　[5] The method for determining the extent of aging according to [4], wherein the blood metabolite comprises at least one metabolite selected from the group consisting of compound B, compound C, compound D, compound F, compound H, compound I, and compound L shown in Table 1.
　[6] The method for determining the extent of aging according to[5], wherein the blood metabolite comprises at least one metabolite selected from the group consisting of compound D, compound F, compound L shown in Table 1.
　[7] An apparatus for determining the extent of aging in which the extent of aging is determined by the method according to any one of [1] to [6].
　[8] An apparatus for determining the extent of aging which comprises means for input and means for determining, wherein data of blood metabolites of the subject are input to the means for input, and the extent of aging is determined by comparing the data of the subject and the data of the population.
　[9] A system for determining the extent of aging in which the extent of aging is determined by the method according to any one of [1] to [6], or the apparatus according to [7] or [8].
　[10] A method of evaluating substances which affect the extent of aging comprising the step of measuring a blood metabolite, wherein the blood metabolite comprises at least one metabolite selected from the group consisting of compound B, compound C, compound D, compound F, compound G, compound H, compound I, compound J and compound L shown in Table 1.

　[11] A kit for determining the extent of aging by using the methods according to any one of [1] to [6], comprising blood collection tubes and blood metabolite compounds as detection standard.
　[12] 2-methoxy-4-(trimethylammonio) butanoate as an indicator for human aging.
　[13] 3-methoxy-4-(trimethylammonio) butanoate as an indicator for human aging.

Human blood provides a rich source of information about metabolite that reflects individual differences in health, disease, diet and life-style. We found novel 9 age-related metabolites, named compounds B-L. While the chemical structure of which were not determined, their molecular formula was determined based on the data obtained by high-resolution mass spectrometry. Moreover, tandem mass analysis narrowed down the candidate structures of compounds B-L. The profiles of these compounds are shown in Table 1 in the specification. These compounds showed more abundance in elderly (>80 yr) blood samples than in youth (<40 yr).

Representative LC-MS chromatograms of human blood sample; Carnitine and compound D, compound B and H, compounds F and I are identical in mass respectively, but their RTs were different. Peak abundance of compounds B-L and carnitine in 15 young and 15 elderly people; Distributions of compound B, compound C, compound D, compound F, compound G, compound H, compound I, compound J, and compound L in blood of 30 individuals. Correlation among compounds B-L and carnitine; Correlation coefficients among compounds B-L are shown. Among Compound B-G and between I and L showed very strong correlation. It suggests these compounds may have close relationship on biosynthetic pathway and similar physiological function. No correlation with carnitine. Peak abundance in RBCs and plasma; RBC/plasma ratio of the compounds are shown. Compounds B-L are enriched in RBC that indicate these are accumulated in RBC. Carnitine had no difference between in RBC and plasma. LC-MS chromatogram and MS2 spectra of 2-methoxy-4-(trimethylammonio)butanoate; The retention time and accurate mass of the peak of a synthetic standard, 2-methoxy-4-(trimethylammonio)butanoate, clearly matched with the peak of compound F in a blood sample. Detected 4 kinds of MS2 fragment also matched with ones of compound F. LC-MS chromatogram and MS2 spectra of 3-methoxy-4-(trimethylammonio)butanoate;The retention time and accurate mass of the peak of a synthetic standard, 3-methoxy-4-(trimethylammonio)butanoate, clearly matched with the peak of compound I in a blood sample. Detected 3 kinds of MS2 fragment also matched with ones of compound I.

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 "extent of aging" is used herein to refer to the degree of aging or aging index. It is a value indicating whether the speed of aging of the subject is earlier or later than the average.

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 determining the extent of aging
According to the present invention, the extent of aging is evaluated by using a specific blood metabolite in a subject as an indicator. By measuring the amount of a specific blood metabolite in whole blood, erythrocytes or plasma of the subject, the extent of senescence (aging degree) of the subject can be determined.

Here, the sample used for determining the aging extent of the 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 elderly and the young age group. The blood metabolite in the present invention comprises at least one metabolite selected from the group consisting of compound B, compound C, compound D, compound F, compound G, compound H, compound I, compound J, and compound L shown in Table 1.

Compound B, compound C, compound D, compound F, compound G, compound H, compound I, compound J, and compound L are higher in elder. Therefore when the content of these compounds is higher than standard, the extent of aging of the subject is judged to be high.

Preferably, the blood metabolite in the present invention comprises at least one metabolite selected from the group consisting of compound B, compound C, compound D, compound F, compound H, compound I, and compound L shown in Table 1.

More preferably, the blood metabolite in the present invention comprises at least one metabolite selected from the group consisting of compound D, compound F, compound L shown in Table 1.

In order to obtain a more accurate determination result of aging extent, it is preferable to analyze plural blood metabolites.

Structures of candidate compound B are shown below.

Structures of candidate compound C are shown below.

Structures of candidate compound D are shown below.

Structures of candidate compound F are shown below. 2-methoxy-4-(trimethylammonio)butanoate which is highlighted by a square is confirmed to be one of compound F.

Structures of candidate compound G are shown below.

Structures of candidate compound H are shown below.

Structures of candidate compound I are shown below. 3-methoxy-4-(trimethylammonio)butanoate which is highlighted by a square is confirmed to be one of compound I

Structures of candidate compound J are shown below.

Structures of candidate compound L are shown below.

The method for determining the extent of aging of the present invention comprises (i) a step of preparing a sample, (ii) a step of analysis and (iii) a step of determining the extent of aging.

(i) a step of preparing a sample
Metabolomic samples can be prepared as reported previously (NPL 13). 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.

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 (7~100×10⁸ RBC), 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. 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 analysis
The content of blood metabolite in the sample of the subject is analyzed in this step. LC-MS data are preferably to be obtained using a UltiMate3000 DGP-3600RS system (Thermo Fisher Scientific, Waltham, MA, USA) coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific), as previously described (NPL 14). Briefly, LC separation is performed on a ZIC-pHILIC column (Merck SeQuant, Umea, Sweden; 150 mm × 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 13). 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. An electrospray ionization (ESI) source was used for MS detection. Each sample was injected twice (1 μl volume / injection); one with the ESI operated in negative ionization mode and the other in positive ionization mode. Spray voltage and capillary temperature were set to 2.5 kV, 299 °C (negative ESI) or 3.5 kV, 299 °C (positive ESI) respectively. Nitrogen was used as the carrier gas. The mass spectrometer was operated in a full scan mode (240,000 resolution) with a 700-1000 m/z scan rage and automatic data-dependent MSn fragmentation scans (15,000 resolution) at collision energy of 40±20% (HCD) or 4% (CID). Peak areas of metabolites of interest are measured using Xcalibur software (Thermo Fisher Scientific) or MZmine 2 software (NPL 15).

(iii) a step of determining the extent of aging
The method for determining the extent of aging of the present invention is not particularly limited as long as it uses the above metabolite as an index. The following method is exemplified as a merely example. The age score (calculated value) can be determined from the data of the aging marker of the subject based on the standard curve made from the plot of the aged marker's quantitative value (peak area) and calendar age. And the extent of aging can be determined by the difference from the calendar age. For example, when dividing the age score of a metabolite by a calendar age and multiplying by 100, the young tendency is judged to be as low as 100 and the older tendency is judged as higher than 100.

Apparatus
The present invention provides an apparatus for determining the extent of aging. The apparatus uses the method of the present invention above.

The apparatus for determining the extent of aging of the present invention comprises means for input and means for determining, wherein data of blood metabolites of the subject are input to the means for input, and the extent of aging is determined by comparing the data of the subject with the data of the population. 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 determining the extent of aging. The extent of aging is determined by the method of the present invention above, or the apparatus of the present invention above. 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 affect the extent of aging comprising the step of measuring a blood metabolite, wherein the blood metabolite comprises at least one metabolite selected from the group consisting of compound B, compound C, compound D, compound F, compound G, compound H, compound I, compound J, and compound L shown in Table 1. The substances found by this evaluation method can be widely used as anti-aging foods, drinks, supplements, pharmaceuticals, cosmetics and the like. The section of "A method for determining the extent of aging" can be referred for details of the step of measuring a blood metabolite.

Kit
The present invention provides a kit for determining the extent of aging 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 compound B, compound C, compound D, compound F, compound G, compound H, compound I, compound J, and compound L shown in Table 1.

Future Prospects of Human Metabolomics.
It could be considered that novel 9 blood metabolites found in the present invention are correlated with aging related diseases. Based on the blood levels of these metabolites as indicators, it may be possible to determine risk of disease, status of disease, susceptibility to disease, etc. Following diseases can be exemplified as said diseases. Lifestyle-related disease (such as atherosclerosis, hypertension, type 2 diabetes mellitus, menopause, osteoporosis, cancer); Neurological disorder (such as brain infarction, Alzheimer disease, dementia, Parkinson Syndrome); Eye disease (such as cataract, glaucoma, age-related macular degeneration, presbyopia, dry eye); Otorhinolaryngologic disease (such as hearing disturbance, chronic thyroiditis, xerostomia); Hematological disorder (such as malignant lymphoma, leukemia, anemia); Heart disease (such as ischemic heart disease, myocardial infarction, heart failure, angina pectoris, acute coronary syndrome); Pulmonary disease (such as COPD (Chronic obstructive pulmonary Disease), lung fibrosis); Digestive disease (such as atrophic gastritis, liver cirrhosis, fatty liver, liver dysfunction); Kidney & urological disease (such as urine incontinence, late onset hypogonadism syndrome, chronic renal failure, prostate hypertrophy), Musculoskeletal disease (such as arthritis, locomotive syndrome, sarcopenia, lumbago, joint pain, frailty), poor nutrition, progeria, Werner syndrome and the like.

We identified human blood metabolites that strikingly differ in abundance between 15 young (<40 yr) and 15 elderly (>80 yr) groups using non-targeted, comprehensive LC-MS (liquid chromatography-mass spectrometry) (Figure 1). P-values from the Mann-Whitney test, for the age differences, were less than 0.01 (Figure 2). Peak areas reflecting the abundance of compounds were high (10⁸) for D , but low (10⁶) for G, H, and J, and the remaining compounds were medium abundance (Table 2). The detected mass (m/z) of compounds B-L was in the range from 146.1174 to 200.1279. The predicted formula consists C_7-10 H_15-17 N₁ O_2-4. MS/MS (MS2) and MS/MS/MS (MS3) fragments of these compounds obtained by Orbitrap Fusion Lumos contained the common fragment (Table 2). Namely, the fragment masses 58.065 (C₃H₈N), 60.081 (C₃H₁₀N), or 74.096 (C₄H₁₂N), presumably derived from N,N,N-trimethylammonium, were present in all compounds. In liquid chromatography (LC), these compounds B-L were eluted at different but close RT (retention time, 8.0~11.5 min). Whilst compounds B and H have the identical mass, their retention time differed significantly. Similarly, compounds F and I are identical in mass, their RTs were different. Compound D and carnitine had the same mass, but their RTs were different (Figure 1, Table 2).

The degree of correlation was examined for these compounds among 30 subjects. As shown in Figure 3, the correlation values were surprisingly high (0.86~0.95) among compounds B, C, D, F and G. However the correlation between compounds B-G and compounds H-L was relatively low (0.46~0.84). It was noticed that carnitine, predicted structural similar to compounds B-L, showed low correlation values respectively, to these compounds.

It was found that compounds B-L were enriched in RBCs. Blood cells were fractionated by RBCs and plasma, and they were analyzed by LC-MS. As shown in Figure 4, the ratio of peak abundance between RBC and plasma was high in the range between 3.4 and 21.9, showing that these compounds were RBCs enriched that indicate these are accumulated in RBC. Carnitine had no difference between in RBC and plasma.

We predicted chemical structure of compounds B-L based on tandem MS data (Table 2). Identification of any peak by tandem MS analysis in the absence of a standard requires the m/z value and fragmentation pattern. A peak with an m/z value of 162.1122 Dalton (Da) in positive ionization mode matched the calculated value for the carnitine (C₇H₁₆O₃N) positive ion with a hydrogen adduct (162.1130 Da). The MS2 fragmentation pattern matches the compound D structure; the 102.091 Da (C₅H₁₂ON) fragment corresponds to loss of C₂H₄O₂, the 60.080 Da fragment (C₃H₁₀N) to loss of C₂H₄O₂ and C₂H₂O. While compound D showed the 130.086 Da fragment (C₆H₁₂O₂N) corresponds to loss of CH₃O, we could not detect the same fragment in carnitine; thus we narrowed 3 kinds of planar structure as candidates of compound D shown below.

A peak with an m/z value of 192.1228 Da in positive ionization mode matched the calculated value for the formula C₈H₁₈O₄N positive ion with a hydrogen adduct (192.1236 Da). From the MS2 and MS3 fragmentation pattern; the 133.049 Da (C₅H₉O₄) fragment corresponds to loss of C₃H₉N, the 73.028 Da fragment (C₃H₅O₂) to loss of C₅H₉O₄ and C₂H₄O₂, and other data; we narrowed 4 kinds of planar structure as candidates of compound B shown below.).

A peak with an m/z value of 174.1121 Da in positive ionization mode matched the calculated value for the formula C₈H₁₆O₃N positive ion with a hydrogen adduct (174.1130 Da). From the MS2 and MS3 fragmentation pattern; the 114.091 Da (C₆H₁₂ON) fragment corresponds to loss of C₂H₄O₂, the 58.065 Da fragment (C₃H₈N) to loss of C₂H₄O₂ and C₃H₄O, and other data; we narrowed 8 kinds of planar structure as candidates of compound C shown below.

A peak with an m/z value of 176.1278 Da in positive ionization mode matched the calculated value for the formula C₈H₁₈O₃N positive ion with a hydrogen adduct (176.1287 Da). From the MS2 and MS3 fragmentation pattern; the 117.054 Da (C₅H₉O₃) fragment corresponds to loss of C₃H₉N, the 89.059 Da fragment (C₄H₉O₂) to loss of C₃H₉N and CO, and other data; we narrowed 6 kinds of planar structure as candidates of compound F shown below. 2-methoxy-4-(trimethylammonio)butanoate which is highlighted by a square is confirmed to be one of compound F.

A peak with an m/z value of 200.1279 Da in positive ionization mode matched the calculated value for the formula C₁₀H₁₈O₃N positive ion with a hydrogen adduct (200.1287 Da). From the MS2 and MS3 fragmentation pattern; the 140.107 Da (C₈H₁₄ON) fragment corresponds to loss of C₂H₄O₂, the 84.080 Da fragment (C₅H₁₀N) to loss of C₂H₄O₂ and C₃H₄O, and other data; we narrowed 4 kinds of planar structure as candidates of compound G shown below.

A peak with an m/z value of 192.1227 Da in positive ionization mode matched the calculated value for the formula C₈H₁₈O₄N positive ion with a hydrogen adduct (192.1236 Da). From the MS2 and MS3 fragmentation pattern; the 146.117 Da (C₇H₁₆O₂N) fragment corresponds to loss of CH₂O₂, the 99.067 Da fragment (C₅H₉ON) to loss of CH₂O₂ and C₂H₇O, and other data; we narrowed 3 kinds of planar structure as candidates of compound H shown below.

A peak with an m/z value of 176.1278 Da in positive ionization mode matched the calculated value for the formula C₈H₁₈O₃N positive ion with a hydrogen adduct (176.1287 Da). From the MS2 and MS3 fragmentation pattern; the 117.054 Da (C₅H₉O₃) fragment corresponds to loss of C₃H₉N, the 85.028 Da fragment (C₄H₅O₂) to loss of C₃H₉N and CH₄O, and other data; we narrowed 9 kinds of planar structure as candidates of compound I shown below. 3-methoxy-4-(trimethylammonio)butanoate, which is highlighted by a square is confirmed to be one of compound I.

A peak with an m/z value of 146.1174 Da in positive ionization mode matched the calculated value for the formula C₇H₁₆O₂N positive ion with a hydrogen adduct (146.1181 Da). From the MS2 fragmentation pattern; the 87.044 Da (C₄H₇O₂) fragment corresponds to loss of C₃H₉N and other data; we narrowed 5 kinds of planar structure as candidates of compound J shown below.

A peak with an m/z value of 160.1334 Da in positive ionization mode matched the calculated value for the formula C₈H₁₈O₂N positive ion with a hydrogen adduct (160.1338 Da). From the MS2 and MS3 fragmentation pattern; the 101.059 Da (C₅H₉O₂) fragment corresponds to loss of C₃H₉N, the 55.054 Da fragment (C₄H₇) to loss of C₃H₉N and CH₂O₂, and other data; we narrowed 9 kinds of planar structure as candidates of compound L shown below.

2-methoxy-4-(trimethylammonio) butanoate or 3-methoxy-4-(trimethylammonio) butanoate are candidates of compound F and I respectively, predicted based on MS/MS fragment data. These compounds are novel and have not been described in any literature. Therefore we investigated the structure of these compounds by comparison analytical data between a blood sample and synthesized standard. As shown in Figure 5, the RT and accurate mass (11.397 and 176.128) of the peak of 2-methoxy-4-(trimethylammonio) butanoate clearly matched with ones (11.441 and 176.128) of the peak of compound F in a blood sample (long arrows in upper panel). Moreover, detected 4 kinds of MS2 fragments also matched with ones of compound F (short arrows in lower panel). Also, as shown in Figure 6, the RT and accurate mass (9.956 and 176.128) of the peak of 3-methoxy-4-(trimethylammonio)butanoate clearly matched with ones (10.045 and 176.128) of the peak of compound I in a blood sample (long arrows in upper panel). Moreover, detected 3 kinds of MS2 fragments also matched with ones of compound I (short arrows in lower panel). These data indicate that these two compounds 2-methoxy-4-(trimethylammonio)butanoate and 3-methoxy-4-(trimethylammonio) butanoate are strong candidates of compounds F and I respectively.

Many of the other candidate compounds were not available because they are technically difficult to synthesize. The chemical structure and physiological significance of these compounds B-L are unknown yet, but monitoring them based on the information provided by the present invention may be helpful for understanding the biological aging of individuals.

Ethics statement
Written, informed consent was obtained from all donors, 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 Human Subjects Research Review Committee of the Okinawa Institute of Science and Technology Graduate University (OIST).

Human subject characteristics and blood metabolomics analysis
30 healthy male and female volunteers participated in this study (Table 3). Metabolomic samples were prepared as reported previously (NPL 13). Blood samples for metabolomics analysis and clinical blood parameters were taken in the morning and subjects were asked not to eat breakfast to ensure at least 12 hr of fasting.

Claims (13)

1. A method for determining the extent of aging in which a blood metabolite is used as an indicator.
2. The method for determining the extent of aging according to claim 1, wherein whole blood or Red blood cells from a subject are used as a sample, and a blood metabolite in the sample is used as an indicator.
3. The method for determining the extent of aging according to claim 2, wherein the sample is treated with cold organic solvent immediately after bleeding.
4. The method for determining the extent of aging according to any one of claims 1 to 3, wherein the blood metabolite comprises at least one metabolite selected from the group consisting of compound B, compound C, compound D, compound F, compound G, compound H, compound I, compound J, and compound L shown in Table 1.
   

   

   
   

   
5. The method for determining the extent of aging according to claim 4, wherein the blood metabolite comprises at least one metabolite selected from the group consisting of compound B, compound C, compound D, compound F, compound H, compound I, and compound L shown in Table 1.
6. The method for determining the extent of aging according to claim 5, wherein the blood metabolite comprises at least one metabolite selected from the group consisting of compound D, compound F, compound L shown in Table 1.
7. An apparatus for determining the extent of aging in which the extent of aging is determined by the method according to any one of claims 1 to 6.
8. An apparatus for determining the extent of aging which comprises means for input and means for determining, wherein data of blood metabolites of the subject are input to the means for input, and the extent of aging is determined by comparing the data of the subject and the data of the population.
9. A system for determining the extent of aging in which the extent of aging is determined by the method according to any one of claims 1 to 6, or the apparatus according to claim 7 or 8.
10. A method of evaluating substances which affect the extent of aging comprising the step of measuring a blood metabolite, wherein the blood metabolite comprises at least one metabolite selected from the group consisting of compound B, compound C, compound D, compound F, compound G, compound H, compound I, compound J, and compound L shown in Table 1.
    

    

    
    

    
11. A kit for determining the extent of aging by using the methods according to any one of claims 1 to 6, comprising blood collection tubes and blood metabolite compounds as detection standard.
12. 2-methoxy-4-(trimethylammonio)butanoate as an indicator for human aging.
13. 3-methoxy-4-(trimethylammonio)butanoate as an indicator for human aging.

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