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. 2013 Apr 23;7:9. doi: 10.3389/fnsys.2013.00009

Cerebral Low-Molecular Metabolites Influenced by Intestinal Microbiota: A Pilot Study

Mitsuharu Matsumoto 1,*, Ryoko Kibe 2, Takushi Ooga 3, Yuji Aiba 4, Emiko Sawaki 1, Yasuhiro Koga 4, Yoshimi Benno 2
PMCID: PMC3632785  PMID: 23630473

Abstract

Recent studies suggest that intestinal microbiota influences gut-brain communication. In this study, we aimed to clarify the influence of intestinal microbiota on cerebral metabolism. We analyzed the cerebral metabolome of germ-free (GF) mice and Ex-GF mice, which were inoculated with suspension of feces obtained from specific pathogen-free mice, using capillary electrophoresis with time-of-flight mass spectrometry (CE-TOFMS). CE-TOFMS identified 196 metabolites from the cerebral metabolome in both GF and Ex-GF mice. The concentrations of 38 metabolites differed significantly (p < 0.05) between GF and Ex-GF mice. Approximately 10 of these metabolites are known to be involved in brain function, whilst the functions of the remainder are unclear. Furthermore, we observed a novel association between cerebral glycolytic metabolism and intestinal microbiota. Our work shows that cerebral metabolites are influenced by normal intestinal microbiota through the microbiota-gut-brain axis, and indicates that normal intestinal microbiota closely connected with brain health and disease, development, attenuation, learning, memory, and behavior.

Keywords: intestinal microbiota, cerebrum, metabolome, gut-brain axis, neurotransmitter

Introduction

Intestinal microbiota play a fundamentally important role in health and diseases (Backhed et al., 2005). Recently, the relationship between intestinal microbiota and systemic phenomena beyond the intestinal environment, such as obesity (Turnbaugh et al., 2006) and lifespan (Matsumoto et al., 2011), have been reported. The bidirectional signaling between the gastrointestinal tract and the brain, the gut-brain axis, is vital for maintaining homeostasis and is regulated at the neural, hormonal, and immunological levels. The importance of the gut-brain axis is further emphasized by the high incidence of co-morbidities between stress-related psychiatric disorders such as anxiety, and gastrointestinal disorders (Camara et al., 2009). Recent studies have investigated the effect of gut microbiota on brain and behavior. The results of these studies suggest that intestinal microbiota have a great impact on gut-brain communication, which led to the coining of the term “microbiota-gut-brain axis” (MGB axis) (Rhee et al., 2009; Cryan and Dinan, 2012). For example, intestinal microbiota modulates brain development and subsequent adult behavior, such as motor activity and anxiety (Heijtz et al., 2011; Neufeld et al., 2011). Studies on the MGB axis have focused on the central nervous system (CNS), including the hypothalamic-pituitary-adrenal axis (Sudo et al., 2004; Rhee et al., 2009), neurotransmitter, and synapse related factors (for example, PSD-95, synaptophysin; Heijtz et al., 2011), and brain-derived neurotrophic factor (Heijtz et al., 2011; Neufeld et al., 2011). However, to the best of our knowledge, other metabolites stimulated by the MGB axis have not been investigated. Furthermore, some metabolites may be synthesized independently in the brain and may be influenced by MGB axis, while some metabolites produced by intestinal bacteria may be transported from the colonic lumen to the brain in the bloodstream without filtration by blood-brain barrier (BBB).

Capillary electrophoresis with time-of-flight mass spectrometry (CE-TOFMS) is a novel strategy for analyzing and differentially displaying metabolic profiles (Monton and Soga, 2007). Here, using CE-TOFMS, we analyzed the cerebral metabolome obtained from germ-free (GF) mice and Ex-GF mice, harboring intestinal microbiota from specific pathogen-free mice and demonstrated the large effect of intestinal microbiota on the cerebral metabolome.

Materials and Methods

Mice

Germ-free BALB/c mice were purchased originally from Japan Clea Inc. (Tokyo, Japan), and were bred in the Department of Infectious Diseases, Tokai University School of Medicine, Kanagawa, Japan. We divided six male mice bred from mating into two groups, GF mice (GF 1–3) and Ex-GF mice (Ex-GF 1–3). Mice were housed in Trexler-Type flexible film plastic isolators with sterilized clean tip (CLEA Japan, Inc., Tokyo) as bedding. They were given sterilized water and sterilized commercial CL-2 pellets, which consisted of moisture (8.5%), crude protein (24.5%), crude fat (8.0%), crude fiber (4.4%), crude ash (8.5%), and nitrogen free extracts (48.2%), corresponding to 344.7 kcal/100 g (CLEA Japan, Inc.), ad libitum. The diet was sterilized with an autoclave (121°C, 30 min). Surveillance for bacterial contamination was performed by periodic bacteriological examination of feces throughout the experiments. Ex-GF mice were inoculated at 4 weeks of age into the stomach by a metal catheter with 0.5 mL of a 10−1 suspension of feces obtained from SPF BALB/c mice. The protocols approved by the Kyodo Milk Animal Use Committee (Permit Number: 2009-02) and all experimental procedures were performed according to the guidelines of the Animal Care Committee of Tokai University.

Specimen preparation and CE-TOFMS

Mice (7-week-old mice) were sacrificed by cervical dislocation. The brain was resected on ice, and prefrontal cortex was sliced between 2.5 and 3.5 mm anterior to bregma within 5 min of sacrifice. Immediately after the sacrifaction, cardiac blood (approximately approximately 100 μl) was collected, and sodium ethylenediamine tetraacetate plasma (final concentration was 0.13%) was prepared by centrifugation for 20 min at 2,300 × g and 4°C. The samples were stored at −80°C until use.

Cardiac plasma (50 μl) and methanol (450 μl) with 50 μM intestinal standard were vortexed. The plasma homogenate served as crude metabolome and was added to chloroform (500 μl) and Milli-Q (200 μl), mixed, and centrifuged (2,300 × g, for 5 min at 4°C). The aqueous layer was centrifugally filtered through a 5-kDa cutoff filter Ultrafree-MC (Millipore). The filtrated solution was dried up and suspended in 25 μL Milli-Q water just before the measurement. The cerebrums were suspended in methanol (500 μl) with 50 μM intestinal standard and vortexed vigorously five times for 60 s with a MicroSmash MS-100R (Tomy Digital Biology Co., Ltd., Tokyo, Japan) at 4,000 rpm. The resulting cerebrum sample served as crude metabolome that subsequently underwent the same treatment as the plasma crude metabolome.

Metabolomics measurement and data processing were performed as described previously with an Agilent Capillary Electrophoresis System (Ooga et al., 2011). The CE-MS system is the Agilent G1600A Capillary Electrophoresis System connected with the Agilent G1969A LC/MSD TOF (Agilent Technologies, Palo Alto, CA, USA).

RNA preparation and quantitative real-time PCR of the cerebrums

Frozen prefrontal cerebrums were processed for total RNA preparation with TaKaRa FastPure RNA Kits (Takara Bio Inc., Otsu, Japan). The quantity, purity, and integrity were confirmed initially by electrophoresis. cDNA for each sample was synthesized using 200 ng total RNA and PrimeScript RT reagent Kits (Takara Bio Inc.). Real-time PCR was performed with a StepOne Real-Time PCR System (Applied Biosystems) with TaqMan Fast Universal PCR Master Mix (Applied Biosystems) using TaqMan probes (hexokinase 1: Mm00439344_m1, phosphofructokinase: Mm00445461_m1, and β-actin: Mm02619580_g1). The comparative delta Ct method was used for normalizations to the housekeeping gene β-actin.

Intestinal bacterial compositions

Bacterial compositions were determined using pyrosequencing system. Bacterial DNA was isolated from colonic content samples of mice. The 16S rRNA was targeted to identify intestinal bacteria and a pair of universal primers; 27f (5 –AGA GTT TGA TCC TGG CTC AG–3) and 350r (5 –CTG CTG CCT CCC GTA G–3) were used for PCR. Amplicons were applied to GS titanium sequencing Kit (Roche Diagnostics) include emulsion PCR and analyzed by Genome sequencer FLX system (Roche Diagnostics). About 18,000–20,500 sequences in each sample were identified. Sequences data were compared with DDBJ database (Blast) and classified by taxonomic categories.

Data analysis and statistics

Clustering analysis in metabolome was processed by MATLAB 2008a (MathWorks, MA, USA). Differences in relative quantity between GF mice and Ex-GF mice were evaluated for individual metabolites by Welch’s t-test.

Results

The difference in cerebral metabolome between GF and Ex-GF mice

When the mice were sacrificed, the body weights of GF mice were between 22 and 24 g and those of Ex-GF mice were between 22 and 25 g. CE-TOFMS identified 196 (120 cations and 76 anions) metabolites from the cerebral metabolome in both of GF and Ex-GF mice. Hierarchical clustering of metabolite patterns is shown in Figure 1A. A remarkable difference was observed in the cerebral metabolome between GF and Ex-GF mice. Of the 196 metabolites in the cerebral metabolome, the concentrations of 23 metabolites were at least 1.6-fold, and/or significantly (p < 0.05) higher, in GF mice than Ex-GF mice (group GF > Ex-GF). A further 15 metabolites were at least 1.6-fold, and significantly (p < 0.05) higher, in Ex-GF mice than GF mice (group GF < Ex-GF), and/or 158 metabolites showed no difference in concentration or incidence between GF and Ex-GF mice (Figure 1B).

Figure 1.

Figure 1

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Difference in the cerebral metabolome between GF mice and Ex-GF mice. (A) Hierarchical clustering showing patterns of metabolites. Red and green indicate high and low concentrations of metabolites, respectively. (B) The number of cerebral metabolites in the group. GF > Ex-GF; GF ≈ Ex-GF; and GF < Ex-GF.

Identified metabolites were classified into eight categories and are listed in Table A1 in Appendix (anion) and Table A2 (cation) in Appendix. Metabolites, in which there are significant differences between GF and Ex-GF mice, are shown in Tables 1 and 2.

Table 1.

Metabolites whose concentrations were higher in the cerebral metabolome of Ex-GF mice than in that of GF mice.

Compound name Category Mean
 SD
 Ratio
GF Ex-GF GF Ex-GF Ex-GF/GF
Trimethylamine N-oxide Alkylamino acid 1.87E-05 8.20E-05 3.37E-06 1.53E-05 4.39 *
N5-Ethylglutamine Alkylamino acid 6.06E-05 1.43E-04 6.62E-06 2.43E-05 2.36 *
Cysteine glutathione disulfide Peptide 3.12E-04 6.78E-04 2.78E-04 4.36E-04 2.17
2,3-Diphosphoglyceric acid 8.67E-05 1.61E-04 2.73E-05 3.98E-05 1.85 p < 0.1
Cys Amino acid 8.61E-04 1.54E-03 7.09E-04 9.51E-04 1.79
2-Methylserine 4.90E-05 8.70E-05 4.25E-06 1.25E-05 1.78 *
3-Methylhistidine Alkylamino acid 6.14E-04 1.03E-03 6.71E-05 1.34E-04 1.68 *
Cystine Peptide 1.96E-05 3.28E-05 NA 5.53E-06 1.67
Trp Amino acid 9.74E-04 1.44E-03 2.74E-05 1.13E-04 1.48 *
Pipecolic acid 1.61E-04 2.33E-04 7.37E-07 1.28E-05 1.44 *
Tyr Amino acid 3.47E-03 4.75E-03 3.16E-04 3.81E-04 1.37 *
Phe Amino acid 3.74E-03 4.97E-03 1.44E-04 1.38E-04 1.33 ***
Asp Amino acid 2.72E-03 3.43E-03 2.78E-05 1.20E-04 1.26 **
Ribose 5-phosphate Energy 1.11E-04 1.32E-04 3.22E-06 9.09E-06 1.19 *
Gln Amino acid 7.12E-03 8.46E-03 1.74E-04 4.57E-04 1.19 *
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*p < 0.05, **p < 0.01, ***p < 0.001 (GF vs. Ex-GF).

These metabolites have significant or more than 1.6-fold difference between GF mice and Ex-GF.

Table 2.

Metabolites whose concentrations were lower in the cerebral metabolome of Ex-GF mice than in that of GF mice.

Compound name Category Mean
 SD
 Ratio
GF Ex-GF GF Ex-GF Ex-GF/GF
N-Acetylneuraminic acid Alkylamino acid 1.03E-03 8.75E-04 4.35E-05 5.59E-05 0.85 *
N-Acetylaspartic acid Neuron transmitter 2.06E-01 1.72E-01 1.04E-02 8.54E-03 0.84 *
Pantothenic acid Co-enzyme 4.37E-04 3.58E-04 2.08E-05 3.18E-05 0.82 *
Biotin Co-enzyme 2.22E-04 1.79E-04 1.66E-05 1.83E-05 0.80 *
Ser Amino acid 1.05E-03 7.95E-04 6.49E-05 5.53E-05 0.76 **
ADP Nucleic acid 5.09E-03 3.85E-03 5.37E-04 1.32E-04 0.76 *
1-Methylnicotinamide Alkylamino acid 5.04E-05 3.71E-05 5.17E-06 5.00E-06 0.74 *
Ser-Glu Peptide 4.69E-05 3.42E-05 5.47E-06 4.85E-06 0.73 *
Succinic acid Energy 2.01E-02 1.45E-02 1.25E-03 1.68E-03 0.72 *
3-Phenylpropionic acid 1.23E-04 8.80E-05 1.30E-05 9.03E-06 0.71 *
Dihydroxyacetone phosphate Energy 2.02E-04 1.40E-04 1.23E-05 2.27E-05 0.69 *
IMP Nucleic acid 2.67E-03 1.84E-03 1.70E-04 2.63E-04 0.69 *
2-Hydroxybutyric acid 8.39E-05 5.77E-05 8.91E-06 8.28E-06 0.69 *
NADP+ Co-enzyme 8.69E-05 5.68E-05 3.79E-06 1.27E-05 0.65 *
Hydroxyproline Amino acid 1.64E-03 1.07E-03 3.18E-05 1.14E-04 0.65 **
NADH Co-enzyme 2.09E-04 1.35E-04 2.26E-05 2.52E-05 0.65 *
3-Phosphoglyceric acid 5.54E-04 3.44E-04 9.54E-05 2.31E-05 0.62 p < 0.1
Glycerol 3-phosphate 3.77E-03 2.31E-03 2.93E-04 1.27E-03 0.61
Glucose 6-phosphate Energy 1.01E-04 6.03E-05 2.05E-05 2.93E-06 0.60 p < 0.1
Fructose 6-phosphate Energy 2.68E-05 1.54E-05 7.92E-06 2.03E-06 0.57
Dopamine Neuron transmitter 5.26E-04 2.85E-04 2.16E-04 5.60E-05 0.54
Fructose 1,6-diphosphate Energy 5.43E-04 2.67E-04 1.52E-04 4.13E-05 0.49 p < 0.1
Taurocholic acid Bile acid 1.51E-04 2.26E-05 3.08E-05 1.17E-05 0.15 **
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*p < 0.05, **p < 0.01, ***p < 0.001 (GF vs. Ex-GF).

These metabolites have significant or more than 1.6-fold difference between GF mice and Ex-GF.

Influence of intestinal microbiota on cerebral glycolytic metabolism

The relative quantities of the annotated metabolites in the principal metabolic pathways are represented as bar graphs (Figure 2). The concentrations of metabolites involved in glycolysis/gluconeogenesis pathways are characteristically higher in GF mice than in Ex-GF mice. Therefore, we focused our work on cerebral glycolytic metabolism (Figure 3A). The concentration of ADP and NADH were significantly (p < 0.05) higher, while there was a tendency for concentrations of ATP, AMP, and NAD+ to be higher in GF mice than Ex-GF mice. The NADH/NAD+ ratio tended to be lower in GF mice than in Ex-GF mice (Figure 3B). There was no difference in the expression of the hexokinase and phosphofructokinase genes, between GF mice and Ex-GF mice (Figure 3C).

Figure 2.

Figure 2

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Differences of cerebral metabolites between GF mice and Ex-GF mice on the principal metabolic pathways. The relative quantities of the annotated metabolites are represented as bar graphs (blue, GF: red, Ex-GF). Metabolites surrounded by blue and red circles are of higher and lower concentrations, respectively, in GF mice than Ex-GF mice. ND, not detected.

Figure 3.

Figure 3

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Comparison of glycolytic metabolic activity between GF mice and Ex-GF mice. (A) The relative quantities of the annotated metabolites are represented as bar graphs (blue, GF: red, Ex-GF). (B) Relative quantities of ATP, ADP, AMP, and nicotinamides (*p < 0.05). (C) Cerebral gene expression of hexokinase and phosphofructokinase. Data are represented as mean ± SD (A,B) and mean ± SEM (C).

Comparison of individual differences in metabolome between colonic luminal content, cardiac plasma, and the cerebrum

Relative standard deviations (RSD% = value of standard deviation/value of mean × 100) of metabolites in the colonic luminal content, cardiac plasma, and the cerebrum of GF and Ex-GF mice are shown in Figure 4. The RSD value of metabolites in the cerebrum was similar between GF and Ex-GF mice. However, in Ex-GF mice, the RSD values in cardiac plasma (p = 0.10) and colonic luminal content (p < 0.001) were larger than in GF mice. In addition, in Ex-GF mice, the RSD values were the highest for colonic content (vs. cardiac plasma, p < 0.01), followed by cardiac plasma (vs. the cerebrum, p < 0.05) and the cerebrum. In contrast, in GF mice, the RSD value did not differ between colonic content and cardiac plasma, although that of cardiac plasma was greater than that of the cerebrum (p < 0.05).

Figure 4.

Figure 4

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The boxplot of RSD% of all metabolites detected from colonic luminal content, cardiac plasma, and the cerebrum. Blue cross bars represent the comparison of means between Ex-GF mice and GF mice. *p < 0.05, **p < 0.01, ***p < 0.001 (GF vs. Ex-GF).

Comparisons of metabolites between colonic luminal content, cardiac plasma, and the cerebrum

We compared the 38 metabolites, which were significantly altered between the cerebrum of GF and Ex-GF mice. The relative quantitative ratio (Ex-GF/GF value) for the expression of each metabolite in colonic luminal content, cardiac plasma, and the cerebrum are shown in Figure 5. Six metabolites, which are shown in red, had similar Ex-GF/GF ratios in all three sites. Although detected in the cerebrum, 12 metabolites, which are shown in blue, were below the detection limit in cardiac plasma. A total of 16 metabolites, which are marked by the “#” symbol, had different Ex-GF/GF ratios between the cerebrum and cardiac plasma. The Ex-GF/GF ratios of all other metabolites did not differ between the three specimens.

Figure 5.

Figure 5

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Relative quantitative ratio (Ex-GF/GF value) comparisons of 38 metabolites between GF mice and Ex-GF mice, colonic luminal content, cardiac plasma, and the cerebrum. Metabolites shown in red have similar Ex-GF/GF ratios between the colonic lumen, cardiac plasma, and the cerebrum. Metabolites shown in blue are below the detection limit in cardiac plasma, but were detected in the cerebrum. #These metabolites differed in Ex-GF/GF ratios between the cerebrum and cardiac plasma. *p < 0.05, **p < 0.01, ***p < 0.001 (GF vs. Ex-GF). ND, not detected.

Intestinal bacterial compositions

Bacterial compositions were analyzed using FLX systems and the results are shown in Figure 6. Phylum Firmicutes (80%) and phylum Bacteroidetes (about 6%) have been identified as dominant populations in all samples. Following detailed classification on the family level, the families Lactobacillaceae, Lachnospiraceae, Clostridiaceae, and Bacteroidaceae were commonly detected and constituted higher proportions in the population, i.e., 50–70, 3–10, 2–5, and 2–4% respectively, than other families. However, there were only small individual differences among the samples. These families accounted for up to 60–70% of the total bacterial population.

Figure 6.

Figure 6

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Aggregate microbiota composition at the phylum and family levels in the colonic content of Ex-GF mice.

Discussion

To the best of our knowledge, in a prior study by Fu et al. (2011), the highest numbers of metabolites from brain tissue to date were detected using GC-MS. In total, 118 metabolites were routinely detected in more than 80% of samples in one or more of three species (human, chimpanzee, or rhesus macaques), in at least one brain region (prefrontal or cerebellar cortex). However, only 61 metabolites were annotated. CE-TOFMS identified 196 metabolites from the cerebral metabolome, indicating that CE-TOFMS is more sensitive than GC-MS for comprehensive and large-scale metabolomic analysis in the brain.

Neurotransmitters and several metabolites which are involved in brain function

Concentration of dopamine (DA), a target for amphetamine stimulation of locomotor activity and stereotyped behaviors, was approximately twofold higher (p = 0.188) in GF mice than in Ex-GF mice. This is consistent with the findings that GF mice display increased motor activity and reduced anxiety compared with their Ex-CF counterparts (Heijtz et al., 2011; Neufeld et al., 2011). It is confusing that the concentration of Tyr in the cerebrum of Ex-GF mice was higher than that of GF mice, since Tyr is a precursor of DA. Tyr hydroxylase hydroxylates Tyr to l-DOPA, which was below the detection limit in this study. DOPA is further converted to DA by aromatic amino acid decarboxylase (Daubner et al., 2011). Therefore, this indicates that cerebral DA synthesis is induced by DA-producing enzymes, which are inhibited by stimulation of intestinal microbiota through the MGB axis (Figure 7A). Parkinson disease is characterized by a progressive loss of dopaminergic neurons in the substantia nigra. Since the activity level of Tyr hydroxylase is associated with Parkinson disease (Haavik and Toska, 1998), it is possible that the intestinal microbiota is involved in the development of Parkinson disease.

Figure 7.

Figure 7

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Relative quantitative comparisons of metabolites in the biosynthetic pathway for dopamine (A) and serotonin (B), in the cerebrum of GF mice and Ex-GF mice. Data are represented as mean ± SD. *p < 0.05, ***p < 0.001 (GF vs. Ex-GF).

We were also surprised to find that the concentrations of Trp, precursors of serotonin (5-HT), in the cerebrum of Ex-GF mice were higher than that of GF mice. This was despite the fact that cerebral 5-HT concentration did not differ between GF mice and Ex-GF mice (Figure 7B). It is believed that brain 5-HT concentration is dependent on the brain Trp level (Fernstrom, 2005). Plasma Trp are transported into the brain by a transporter, located at BBB on CNS capillary endothelial cells (Pardridge, 1998), and converted to 5-HT in neurons containing Trp hydroxylase, the rate-limiting enzyme in 5-HT synthesis (Jequier et al., 1967). Therefore, we suppose that cerebral 5-HT synthesis is regulated by Trp hydroxylase in neurons without the influence of the cerebral Trp pool and/or intestinal microbiota under the non-stressed condition and in non-neonates, as in our present study.

Several metabolites, which are known to be involved in brain function, are also influenced by normal intestinal microbiota. N-acetylaspartic acid (NAA), which is in group GF > Ex-GF, is an amino acid present in the vertebrate brain that is synthesized and stored primarily in neurons and considered a marker for neuronal health and attenuation (Simmons et al., 1991; Jenkins et al., 2000). Pipecolic acid, which is in the GF < Ex-GF group, is known as a neuromodulator or neurotransmitter with the gamma-aminobutyric acid (GABA)ergic transmission. Pipecolic acid was shown to be region- and site-specific in the CNS (Kase et al., 1980), which causes hepatic encephalopathy by inducing neuronal cell death, or apoptosis, rather than by depressing neurotransmissions (Matsumoto et al., 2003). Ser was in the GF > Ex-GF group; d-Ser is synthesized from l-Ser by serine racemase (CE-TOFMS could not separate d-Ser and l-Ser) in the human brain. It functions as an obligatory co-agonist at the glycine modulatory site of N-methyl-d-aspartate (NMDA)-selective glutamate receptors. Thus, depletion of d-Ser levels has been implicated in NMDA receptor hypofunction, which is thought to occur in schizophrenia (Yang et al., 2010). N-acetylneuraminic acid (NANA), which was in group GF > Ex-GF, increased learning and memory performance (Wang et al., 2007). These findings indicate that intestinal microbiota are closely related to brain health, disease development, attenuation, learning, and memory.

Cerebral energy metabolism

The concentration of several cerebral glycolysis intermediates was higher in GF mice than in Ex-GF mice (Figure 3A). This raises the following two possibilities: first, the cerebral energy consumption of Ex-GF mice is higher than that of GF mice, and second, that cerebral energy production by glycolysis in Ex-GF mice is lower than in GF mice. However, these phenomena presumably indicate an accelerated molecular flux of the glycolysis pathway to compensate for ATP and NADH depletion in the cerebrum of Ex-GF mice. This assumption is based on our finding that the cerebral ATP (Ex-GF/GF ratio = 0.91) and NADH (Ex-GF/GF ratio = 0.65) levels were lower in Ex-GF mice than GF mice (Figure 3B) and there was no difference in cerebral hexokinase and phosphofructokinase gene expression between mice (Figure 3C). In fact, levels of acetyl CoA, which is produced by oxidation from pyruvic acid, was similar in the cerebrum of GF and Ex-GF mice. Furthermore, a significant difference in lactic acid was not observed, suggesting that the normal intestinal microbiota do not influence anaerobic respiration and the compensated molecular components (ATP or NADH) of the glycolysis pathway in Ex-GF mice was then transferred into the TCA cycle for further aerobic respiration via acetyl CoA in the cerebral mitochondria. To support the presence of an active TCA cycle, we also report changes in NADH and NAD+. The ratio between NADH and NAD+ affects mitochondrial TCA cycle activity (LaNoue et al., 1972). NADH and NADH/NAD+ ratio in Ex-GF mice were reduced to 65 and 92% of those in GF mice, respectively. Since both values are known to increase when the TCA cycle is blocked (Sugiura et al., 2011), the observed reductions in NADH and NADH/NAD+ ratio suggest normal intestinal microbiota induces active oxidative phosphorylation via the TCA cycle. From these findings, we suggest that, in the cerebrum, Ex-GF mice consume energy and accelerate energy production through glycolysis and TCA cycle more highly than GF mice. In other word, the cerebrum of Ex-GF mice is more active than that of GF mice.

Bacterial potential influence on cerebrum metabolic changes

Of 38 metabolites influenced by intestinal microbiota, 12 metabolites detected from the cerebrum but not cardiac plasma, are synthesized independently in the cerebrum and are influenced by MGB axis (Figure 5, metabolites shown in blue). Sixteen metabolites whose Ex-GF/GF ratio differed between the cerebrum and cardiac plasma are influenced by MGB axis and/or BBB (Figure 5, metabolites marked by #). The fact that NANA is in this group is in conflict existing literature. In animal infant models, exogenous administration of NANA increased learning and memory performance as well as the concentration of NANA in the frontal cortex (Carlson and House, 1986; Wang et al., 2007). However, in the present study, NANA produced by intestinal microbiota was not transported to the blood. Therefore, it is doubtful whether dietary NANA influences the brain and behavior directly. We suppose that improvement of learning and memory performance by oral administration of NANA depends on the stimulation of intestinal microbiota, which is altered by supplements containing NANA through the MBG axis. Cerebral GABA concentration did not differ between GF mice and Ex-GF mice, although remarkable differences were observed in GABA cardiac plasma concentrations between GF mice and Ex-GF mice (Figure 8). This indicates that GABA is controlled by BBB and tightly regulated in the cerebrum. This questions the suitability of oral GABA supplementation studies to provide GABA to the brain.

Figure 8.

Figure 8

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Relative quantitative comparisons of GABA in colonic lumen content, cardiac plasma, and the cerebrum. Data are represented as mean ± SD. **p < 0.01.

Differences in RSD values between GF mice and Ex-GF mice (Figure 4) implies that individual differences in the metabolites found in the colonic content and cardiac plasma of Ex-GF mice is influenced by the diversity of intestinal microbiota (Matsumoto et al., 2012). Furthermore, these findings indicate that many metabolites produced by intestinal bacteria are filtrated and transported to brain via the blood through the BBB. However, six cerebral metabolites (Figure 5, metabolites shown in red) had similar Ex-GF/GF ratios between colonic luminal content, cardiac plasma, and the cerebrum. This may suggest that these metabolites may be transported from the colonic lumen to the cerebrum in the bloodstream without filtration by BBB. Further studies are required to fully understand how these metabolites are transported from the gut lumen to blood and from blood to the brain. Furthermore, the relationship between intestinal bacterial composition and brain metabolome is an area that clearly merits further study in the future.

These discussions center on a comparison between general knowledge and the data obtained in the present study. However, the neuronal effects of almost detected metabolites in the cerebrum are unclear. In future studies, researchers in various fields may find evidence that some of the newly identified metabolites are important for neuronal activities and diseases. Indeed, there is a possibility of detecting site-specific metabolome profiles when using CE-TOFMS. Further studies are required to analyze other parts of the brain.

In this study, many metabolites including neurotransmitters showed differences in the concentrations between GF mice and Ex-GF mice, indicating that normal intestinal microbiota closely connected with brain health and disease, development, attenuation, learning, memory, and behavior. We propose that through proper control of intestinal microbiota, cerebral nerve disorders may be prevented or alleviated in the future.

Conflict of Interest Statement

This work was supported by the BRAIN, Japan. This work was also funded by Kyodo Milk Industry Co. Ltd and Human Metabolome Technologies, Inc. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Mitsuharu Matsumoto and Emiko Sawaki are employes of Kyodo Milk Industry Co. Ltd. and had a role in study design, data analysis, preparation of the manuscript, and decision to publish the manuscript. Takushi Ooga is employe of Human Metabolome Technologies, Inc. and had a role in data analysis and decision to publish the manuscript. All of the other authors declare that they have no conflict of interest.

Author Contributions

Mitsuharu Matsumoto wrote the paper. Mitsuharu Matsumoto, Yasuhiro Koga, and Yoshimi Benno designed this study. Yuji Aiba performed animal experiments. Takushi Ooga analyzed the metabolome. Mitsuharu Matsumoto, Ryoko Kibe, Takushi Ooga, and Emiko Sawaki analyzed the data, discussed findings, and helped draft the manuscript.

Acknowledgments

This study was supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry by the Bio-oriented Technology Research Advancement Institution (BRAIN), Japan. We thank Dr. Ayano Yamashita for analyses of gene expression of cerebral glycolytic metabolism.

Appendix

Table A1.

Anionic metabolites detected from cerebrum in GF mice and Ex-GF mice.

ID HMT DB†
 Relative area§
 Comparative analysis
Compound name KEGG ID HMDB ID Germ-free
 SPF
 Germ-free
 SPF
 SPF vs. Germ-free
GF1 GF2 GF3 Ex-GF1 Ex-GF2 Ex-GF3 Mean SD Mean SD Ratio1 p-Value2
A_0052 2,3-Diphosphoglyceric acid C01159 HMDB01294 7.6E-05 6.7E-05 1.2E-04 1.5E-04 1.3E-04 2.0E-04 8.7E-05 2.7E-05 1.6E-04 4.0E-05 1.853 0.064
A_0007 2-Hydroxyisobutyric acid – HMDB00729 1.3E-04 1.6E-04 1.8E-04 3.0E-04 2.2E-04 2.1E-04 1.6E-04 2.3E-05 2.4E-04 4.7E-05 1.525 0.073
A_0041 Phosphocreatine C02305 HMDB01511 1.1E-04 1.1E-04 9.9E-05 1.8E-04 1.6E-04 1.1E-04 1.1E-04 7.2E-06 1.5E-04 3.5E-05 1.430 0.146
A_0015 5-Oxoproline C01879 HMDB00267 1.9E-03 1.0E-03 5.4E-04 2.5E-03 1.6E-03 7.5E-04 1.1E-03 6.9E-04 1.6E-03 8.6E-04 1.398 0.517
A_0088 ADP-ribose C00301 HMDB01178 2.2E-04 2.3E-04 3.8E-04 2.6E-04 3.8E-04 4.1E-04 2.7E-04 9.0E-05 3.5E-04 7.8E-05 1.272 0.338
A_0044 Ribose 5-phosphate C00117 HMDB01548 1.1E-04 1.1E-04 1.1E-04 1.3E-04 1.4E-04 1.2E-04 1.1E-04 3.2E-06 1.3E-04 9.1E-06 1.190 0.044 *
A_0037 Isocitric acid C00311 HMDB00193 1.1E-04 1.7E-04 1.2E-04 1.3E-04 1.5E-04 1.9E-04 1.3E-04 3.1E-05 1.6E-04 3.1E-05 1.189 0.377
A_0087 GTP C00044 HMDB01273 4.1E-04 3.9E-04 5.3E-04 6.2E-04 4.7E-04 4.6E-04 4.5E-04 7.9E-05 5.2E-04 9.0E-05 1.167 0.345
A_0067 NADPH_divalent C00005 HMDB00221 5.4E-05 7.7E-05 7.4E-05 8.5E-05 6.9E-05 8.5E-05 6.8E-05 1.2E-05 8.0E-05 9.4E-06 1.166 0.281
A_0013 2-Hydroxyvaleric acid – HMDB01863 1.5E-04 1.7E-04 1.1E-04 1.9E-04 1.3E-04 1.9E-04 1.5E-04 2.8E-05 1.7E-04 3.3E-05 1.162 0.397
A_0036 Citric acid C00158 HMDB00094 6.3E-03 7.2E-03 6.3E-03 9.3E-03 5.7E-03 7.8E-03 6.6E-03 5.3E-04 7.6E-03 1.8E-03 1.153 0.433
A_0032 Homovanillic acid C05582 HMDB00118 9.4E-05 1.6E-04 1.6E-04 1.5E-04 1.6E-04 1.5E-04 1.4E-04 3.8E-05 1.5E-04 5.1E-06 1.109 0.568
A_0048 myo-Inositol 1-phosphate myo-Inositol 3-phosphate C01177
C04006 		HMDB00213
HMDB06814		 6.6E-04 6.0E-04 5.5E-04 7.3E-04 5.9E-04 6.7E-04 6.0E-04 5.8E-05 6.6E-04 6.7E-05 1.098 0.317
A_0025 Uric acid C00366 HMDB00289 6.0E-05 7.8E-05 5.9E-05 6.3E-05 7.8E-05 6.9E-05 6.6E-05 1.1E-05 7.0E-05 7.9E-06 1.068 0.592
A_0074 Acetyl CoA_divalent C00024 HMDB01206 5.5E-05 5.4E-05 4.6E-05 4.6E-05 5.6E-05 6.1E-05 5.2E-05 5.3E-06 5.4E-05 7.9E-06 1.048 0.677
A_0023 3-Hydroxy-3-methylglutaric acid C03761 - 3.5E-04 3.1E-04 3.0E-04 3.6E-04 3.4E-04 3.1E-04 3.2E-04 2.3E-05 3.4E-04 2.4E-05 1.047 0.479
A_0077 GDP C00035 HMDB01201 1.5E-03 1.7E-03 2.1E-03 1.9E-03 1.6E-03 2.0E-03 1.8E-03 3.1E-04 1.8E-03 2.3E-04 1.033 0.803
A_0093 UDP-N-acetyl glucosamine C00043 HMDB00290 9.1E-04 1.0E-03 1.0E-03 1.1E-03 9.5E-04 9.9E-04 9.8E-04 6.5E-05 1.0E-03 8.3E-05 1.030 0.654
A_0056 N-Acetyl glucosamine 6-phosphate C00357 HMDB01062 1.2E-04 9.6E-05 8.0E-05 1.3E-04 9.3E-05 7.4E-05 9.7E-05 1.8E-05 1.0E-04 3.0E-05 1.025 0.909
A_0083 CDP-choline C00307 HMDB01413 2.0E-04 1.8E-04 2.6E-04 2.2E-04 1.8E-04 2.4E-04 2.1E-04 4.0E-05 2.1E-04 3.1E-05 1.012 0.935
A_0002 Propionic acid C00163 HMDB00237 1.7E-04 9.9E-05 1.1E-04 1.4E-04 1.3E-04 1.0E-04 1.3E-04 4.0E-05 1.3E-04 2.1E-05 1.010 0.964
A_0092 GDP-mannose GDP-galactose C00096
C02280 		HMDB01163 - 1.8E-04 2.2E-04 2.2E-04 2.1E-04 1.9E-04 2.4E-04 2.1E-04 2.3E-05 2.1E-04 2.4E-05 1.005 0.955
A_0017 Malic acid C00149, C00497, C00711 HMDB00156, HMDB00744 1.5E-02 1.6E-02 1.5E-02 1.5E-02 1.3E-02 1.7E-02 1.5E-02 6.0E-04 1.5E-02 1.8E-03 1.001 0.992
A_0010 Fumaric acid C00122 HMDB00134 1.9E-03 2.1E-03 1.9E-03 1.8E-03 1.7E-03 2.4E-03 2.0E-03 1.0E-04 2.0E-03 3.5E-04 0.997 0.977
A_0016 N-Acetyl-β-alanine C01073 – 5.8E-05 7.2E-05 6.8E-05 6.8E-05 6.1E-05 6.6E-05 6.6E-05 7.3E-06 6.5E-05 3.7E-06 0.987 0.870
A_0091 ADP-glucose GDP-fucose C00498
C00325 		HMDB06557
HMDB01095		 8.2E-05 8.5E-05 8.9E-05 9.7E-05 7.9E-05 7.8E-05 8.6E-05 3.6E-06 8.5E-05 1.0E-05 0.987 0.873
A_0035 N-Acetyl glutamic acid C00624 HMDB01138 7.1E-04 6.6E-04 6.4E-04 6.9E-04 6.6E-04 6.1E-04 6.7E-04 3.6E-05 6.5E-04 4.1E-05 0.981 0.714
A_0029 cis-Aconitic acid C00417 HMDB00072 1.6E-04 2.8E-04 1.5E-04 1.6E-04 1.9E-04 2.3E-04 2.0E-04 7.3E-05 1.9E-04 3.3E-05 0.974 0.920
A_0043 Ribulose 5-phosphate C00199, C01101 HMDB00618 2.5E-03 2.1E-03 1.7E-03 2.1E-03 2.3E-03 1.8E-03 2.1E-03 3.9E-04 2.1E-03 2.4E-04 0.969 0.817
A_0068 CoA_divalent C00010 HMDB01423 4.1E-04 4.1E-04 4.4E-04 3.8E-04 3.8E-04 4.1E-04 4.2E-04 2.0E-05 3.9E-04 2.0E-05 0.927 0.138
A_0022 Pelargonic acid C01601 HMDB00847 8.7E-05 5.5E-05 6.7E-05 5.3E-05 5.4E-05 8.7E-05 7.0E-05 1.6E-05 6.5E-05 1.9E-05 0.927 0.743
A_0006 Lactic acid C00186, C00256, C01432 HMDB00190, HMDB01311 2.9E-01 2.8E-01 3.1E-01 2.7E-01 2.5E-01 3.0E-01 3.0E-01 1.3E-02 2.7E-01 2.7E-02 0.920 0.262
A_0094 CMP-N-acetylneuraminate C00128 HMDB01176 4.7E-04 5.7E-04 5.2E-04 4.9E-04 5.4E-04 3.9E-04 5.2E-04 4.9E-05 4.7E-04 7.7E-05 0.919 0.481
A_0031 Ascorbic acid C00072 HMDB00044 3.5E-02 3.5E-02 3.2E-02 3.3E-02 2.8E-02 3.3E-02 3.4E-02 1.6E-03 3.1E-02 3.0E-03 0.918 0.255
A_0061 cAMP C00575 HMDB00058 2.7E-05 4.3E-05 3.5E-05 3.9E-05 2.3E-05 3.4E-05 3.5E-05 7.8E-06 3.2E-05 7.9E-06 0.916 0.667
A_0085 ATP C00002 HMDB00538 1.1E-03 1.1E-03 1.0E-03 1.1E-03 1.0E-03 7.9E-04 1.1E-03 3.4E-05 9.7E-04 1.7E-04 0.915 0.455
A_0070 FAD_divalent C00016 HMDB01248 5.9E-05 7.3E-05 7.6E-05 6.5E-05 6.3E-05 5.7E-05 6.9E-05 9.2E-06 6.2E-05 4.0E-06 0.890 0.288
A_0090 UDP-glucuronic acid C00167 HMDB00935 1.2E-04 1.4E-04 1.2E-04 1.1E-04 1.1E-04 1.2E-04 1.2E-04 1.3E-05 1.1E-04 4.9E-06 0.879 0.172
A_0051 Glucose 1-phosphate C00103 HMDB01586 1.5E-04 2.0E-04 2.0E-04 1.8E-04 1.3E-04 1.7E-04 1.8E-04 2.9E-05 1.6E-04 2.3E-05 0.869 0.328
A_0018 Threonic acid C01620 HMDB00943 1.2E-03 1.4E-03 1.3E-03 1.3E-03 1.0E-03 1.1E-03 1.3E-03 8.1E-05 1.1E-03 1.8E-04 0.860 0.211
A_0057 N-Acetylneuraminic acid C00270 HMDB00230 1.1E-03 1.0E-03 1.0E-03 9.2E-04 8.9E-04 8.1E-04 1.0E-03 4.3E-05 8.7E-04 5.6E-05 0.846 0.020 *
A_0014 Isethionic acid C05123 HMDB03903 8.2E-04 9.5E-04 1.1E-03 7.7E-04 8.8E-04 7.6E-04 9.5E-04 1.3E-04 8.0E-04 6.6E-05 0.846 0.172
A_0089 UDP-glucose UDP-galactose C00029
C00052 		HMDB00286
HMDB00302		 1.2E-03 1.5E-03 1.3E-03 1.0E-03 1.1E-03 1.3E-03 1.3E-03 1.3E-04 1.1E-03 1.1E-04 0.846 0.110
A_0030 N-Acetylaspartic acid C01042 HMDB00812 2.0E-01 2.0E-01 2.2E-01 1.8E-01 1.6E-01 1.7E-01 2.1E-01 1.0E-02 1.7E-01 8.5E-03 0.838 0.014 *
A_0073 UDP C00015 HMDB00295 1.0E-04 1.2E-04 1.2E-04 1.1E-04 8.5E-05 9.2E-05 1.2E-04 1.1E-05 9.6E-05 1.4E-05 0.833 0.135
A_0009 3-Hydroxybutyric acid C01089, C03197 HMDB00011, HMDB00357, HMDB00442 9.8E-04 1.2E-03 8.5E-04 7.7E-04 8.8E-04 8.4E-04 1.0E-03 1.6E-04 8.3E-04 5.6E-05 0.833 0.196
A_0054 Sedoheptulose 7-phosphate C05382 HMDB01068 9.5E-05 1.1E-04 1.2E-04 6.6E-05 1.0E-04 1.1E-04 1.1E-04 1.4E-05 9.1E-05 2.2E-05 0.831 0.298
A_0042 Pantothenic acid C00864 HMDB00210 4.1E-04 4.5E-04 4.5E-04 3.2E-04 3.7E-04 3.8E-04 4.4E-04 2.1E-05 3.6E-04 3.2E-05 0.819 0.029 *
A_0038 Gluconic acid C00257 HMDB00625 1.3E-04 1.1E-04 1.4E-04 1.1E-04 1.0E-04 9.8E-05 1.3E-04 1.3E-05 1.0E-04 6.6E-06 0.816 0.063
A_0040 Lauric acid C02679 HMDB00638 1.5E-04 1.2E-04 1.5E-04 1.5E-04 1.1E-04 8.5E-05 1.4E-04 1.6E-05 1.1E-04 3.2E-05 0.816 0.308
A_0064 AMP C00020 HMDB00045 1.6E-02 2.1E-02 2.3E-02 1.7E-02 1.3E-02 1.9E-02 2.0E-02 3.7E-03 1.6E-02 3.3E-03 0.810 0.248
A_0046 Biotin C00120 HMDB00030 2.1E-04 2.4E-04 2.2E-04 1.7E-04 2.0E-04 1.6E-04 2.2E-04 1.7E-05 1.8E-04 1.8E-05 0.804 0.038 *
A_0055 N-Acetylglucosamine 1-phosphate C04256 HMDB01367 1.3E-04 1.2E-04 1.2E-04 1.2E-04 9.2E-05 9.0E-05 1.3E-04 6.0E-06 1.0E-04 1.9E-05 0.803 0.147
A_0050 myo-Inositol 2-phosphate – – 7.5E-04 8.0E-04 9.6E-04 5.4E-04 6.3E-04 8.3E-04 8.4E-04 1.1E-04 6.7E-04 1.5E-04 0.799 0.191
A_0045 2-Deoxyglucose 6-phosphate C06369 – 8.4E-05 1.3E-04 6.9E-05 9.6E-05 5.2E-05 7.6E-05 9.4E-05 3.1E-05 7.4E-05 2.2E-05 0.793 0.434
A_0005 Butyric acid C00246 HMDB00039 3.9E-05 5.5E-05 7.0E-05 4.6E-05 5.0E-05 3.4E-05 5.5E-05 1.5E-05 4.3E-05 8.2E-06 0.789 0.333
A_0059 CMP C00055 HMDB00095 2.1E-04 3.5E-04 3.3E-04 2.1E-04 1.7E-04 3.0E-04 3.0E-04 7.6E-05 2.3E-04 6.8E-05 0.757 0.286
A_0076 ADP C00008 HMDB01341 4.7E-03 5.7E-03 4.9E-03 3.8E-03 3.7E-03 4.0E-03 5.1E-03 5.4E-04 3.8E-03 1.3E-04 0.755 0.050 *
A_0078 Adenylosuccinic acid C03794 HMDB00536 6.4E-04 8.0E-04 7.4E-04 4.6E-04 4.7E-04 7.1E-04 7.2E-04 8.1E-05 5.4E-04 1.4E-04 0.753 0.153
A_0019 Ethanolamine phosphate C00346 HMDB00224 1.3E-02 1.6E-02 1.8E-02 1.2E-02 1.1E-02 1.2E-02 1.6E-02 2.3E-03 1.2E-02 3.9E-04 0.743 0.091
A_0066 GMP C00144 HMDB01397 2.5E-03 3.3E-03 3.3E-03 2.1E-03 2.0E-03 2.5E-03 3.0E-03 4.8E-04 2.2E-03 2.6E-04 0.730 0.079
A_0012 Succinic acid C00042 HMDB00254 2.0E-02 2.1E-02 1.9E-02 1.5E-02 1.6E-02 1.3E-02 2.0E-02 1.3E-03 1.4E-02 1.7E-03 0.719 0.012 *
A_0021 3-Phenylpropionic acid C05629 HMDB00764 1.3E-04 1.3E-04 1.1E-04 8.6E-05 8.0E-05 9.8E-05 1.2E-04 1.3E-05 8.8E-05 9.0E-06 0.715 0.023 *
A_0095 NAD+ C00003 HMDB00902 1.3E-03 1.9E-03 1.7E-03 1.2E-03 9.6E-04 1.2E-03 1.6E-03 3.2E-04 1.1E-03 1.5E-04 0.702 0.107
A_0027 Dihydroxyacetone phosphate C00111 HMDB01473 1.9E-04 2.0E-04 2.2E-04 1.2E-04 1.7E-04 1.3E-04 2.0E-04 1.2E-05 1.4E-04 2.3E-05 0.694 0.024 *
A_0065 IMP C00130 HMDB00175 2.5E-03 2.8E-03 2.7E-03 2.1E-03 1.7E-03 1.7E-03 2.7E-03 1.7E-04 1.8E-03 2.6E-04 0.691 0.015 *
A_0008 2-Hydroxybutyric acid C05984 HMDB00008 7.5E-05 8.4E-05 9.3E-05 5.1E-05 6.7E-05 5.6E-05 8.4E-05 8.9E-06 5.8E-05 8.3E-06 0.688 0.021 *
A_0097 NADP+ C00006 HMDB00217 8.3E-05 9.0E-05 8.9E-05 7.0E-05 4.4E-05 5.7E-05 8.7E-05 3.8E-06 5.7E-05 1.3E-05 0.654 0.045 *
A_0096 NADH C00004 HMDB01487 1.9E-04 2.1E-04 2.3E-04 1.5E-04 1.5E-04 1.1E-04 2.1E-04 2.3E-05 1.3E-04 2.5E-05 0.646 0.020 *
A_0060 UMP C00105 HMDB00288 5.4E-04 1.1E-03 1.1E-03 5.4E-04 4.6E-04 7.5E-04 9.1E-04 3.3E-04 5.8E-04 1.5E-04 0.641 0.220
A_0034 3-Phosphoglyceric acid C00197 HMDB00807 6.6E-04 5.3E-04 4.7E-04 3.3E-04 3.7E-04 3.3E-04 5.5E-04 9.5E-05 3.4E-04 2.3E-05 0.620 0.055
A_0028 Glycerol 3-phosphate C00093 HMDB00126 4.0E-03 3.4E-03 3.9E-03 2.4E-03 3.5E-03 1.0E-03 3.8E-03 2.9E-04 2.3E-03 1.3E-03 0.612 0.178
A_0047 Glucose 6-phosphate C00668, C01172, C00092 HMDB01401 8.0E-05 1.0E-04 1.2E-04 5.8E-05 5.9E-05 6.4E-05 1.0E-04 2.0E-05 6.0E-05 2.9E-06 0.598 0.072
A_0049 Fructose 6-phosphate C00085 HMDB00124 2.8E-05 1.8E-05 3.4E-05 1.7E-05 1.6E-05 1.3E-05 2.7E-05 7.9E-06 1.5E-05 2.0E-06 0.574 0.122
A_0062 Fructose 1,6-diphosphate C00354 HMDB01058 4.7E-04 4.4E-04 7.2E-04 2.7E-04 3.0E-04 2.2E-04 5.4E-04 1.5E-04 2.7E-04 4.1E-05 0.491 0.079
A_0086 Taurocholic acid C05122 HMDB00036 1.2E-04 1.7E-04 1.6E-04 3.1E-05 1.4E-05 ND 1.5E-04 3.1E-05 2.3E-05 1.2E-05 0.150 0.010 **
Open in a new tab

1Ex-GF/GF ratio.

2Welch’s t-test (*p < 0.05, **p < 0.01).

ND, not detected.

Table A2.

Cationic metabolites detected from cerebrum in GF mice and Ex-GF mice.

ID HMT DB†
 Relative area of standard
 Comparative analysis
Compound name KEGG ID HMDB ID GF
 Ex-GF
 GF
 Ex-GF
GF1 GF2 GF3 Ex-GF1 Ex-GF2 Ex-GF3 Mean SD Mean SD Ratio1 p-Value2
C_0003 Trimethylamine N-oxide C01104 HMDB00925 2.2E-05 1.6E-05 1.8E-05 8.9E-05 9.3E-05 6.4E-05 1.9E-05 3.4E-06 8.2E-05 1.5E-05 4.390 0.015 *
C_0074 N5-Ethylglutamine C01047 – 6.8E-05 5.5E-05 5.9E-05 1.6E-04 1.2E-04 1.5E-04 6.1E-05 6.6E-06 1.4E-04 2.4E-05 2.361 0.022 *
C_0123 Cysteine glutathione disulfide – HMDB00656 6.3E-04 2.1E-04 9.6E-05 8.1E-04 1.0E-03 1.9E-04 3.1E-04 2.8E-04 6.8E-04 4.4E-04 2.173 0.299
C_0031 Cys C00097, C00736, C00793 HMDB00574, HMDB03417 1.7E-03 5.6E-04 3.6E-04 2.6E-03 1.3E-03 7.3E-04 8.6E-04 7.1E-04 1.5E-03 9.5E-04 1.790 0.381
C_0029 2-Methylserine C02115 – 4.4E-05 5.1E-05 5.2E-05 1.0E-04 7.7E-05 8.3E-05 4.9E-05 4.2E-06 8.7E-05 1.2E-05 1.775 0.025 *
C_0073 3-Methylhistidine C01152 HMDB00479 5.4E-04 6.6E-04 6.5E-04 1.2E-03 9.5E-04 9.5E-04 6.1E-04 6.7E-05 1.0E-03 1.3E-04 1.677 0.018 *
C_0099 Cystine C00491, C01420 HMDB00192 2.0E-05 ND ND 2.9E-05 3.7E-05 ND 2.0E-05 NA 3.3E-05 5.5E-06 1.673 NA
C_0018 Hypotaurine C00519 HMDB00965 6.5E-04 4.2E-04 4.9E-04 9.5E-04 6.9E-04 7.4E-04 5.2E-04 1.2E-04 7.9E-04 1.4E-04 1.524 0.059
C_0089 Trp C00078, C00525, C00806 HMDB00929 1.0E-03 9.8E-04 9.4E-04 1.3E-03 1.5E-03 1.5E-03 9.7E-04 2.7E-05 1.4E-03 1.1E-04 1.480 0.015 *
C_0035 Pipecolic acid C00408 HMDB00070, HMDB00716, HMDB05960 1.6E-04 1.6E-04 1.6E-04 2.2E-04 2.5E-04 2.3E-04 1.6E-04 7.4E-07 2.3E-04 1.3E-05 1.444 0.010 *
C_0044 Thiaproline – – 1.0E-03 5.9E-04 2.8E-04 1.1E-03 9.6E-04 5.4E-04 6.2E-04 3.6E-04 8.7E-04 3.0E-04 1.405 0.410
C_0032 Nicotinamide C00153 HMDB01406 7.3E-03 4.6E-03 5.5E-03 7.7E-03 8.9E-03 7.6E-03 5.8E-03 1.4E-03 8.0E-03 7.4E-04 1.385 0.093
C_0109 Inosine C00294 HMDB00195 1.5E-02 8.3E-03 6.6E-03 1.6E-02 1.7E-02 8.4E-03 1.0E-02 4.5E-03 1.4E-02 4.7E-03 1.378 0.371
C_0079 Tyr C00082, C01536, C06420 HMDB00158 3.3E-03 3.3E-03 3.8E-03 4.3E-03 5.0E-03 4.9E-03 3.5E-03 3.2E-04 4.8E-03 3.8E-04 1.371 0.012 *
C_0060 Threo-β-Methyl aspartic acid C03618 - 9.4E-05 6.7E-05 6.6E-05 9.5E-05 1.1E-04 1.0E-04 7.6E-05 1.6E-05 1.0E-04 6.5E-06 1.339 0.091
C_0072 Phe C00079, C02057, C02265 HMDB00159 3.6E-03 3.9E-03 3.7E-03 4.9E-03 5.1E-03 4.9E-03 3.7E-03 1.4E-04 5.0E-03 1.4E-04 1.330 0.000 ***
C_0053 1-Methyl-4-imidazoleacetic acid C05828 HMDB02820 9.5E-05 7.5E-05 1.0E-04 1.1E-04 1.5E-04 9.5E-05 9.1E-05 1.5E-05 1.2E-04 2.8E-05 1.319 0.209
C_0047 Hypoxanthine C00262 HMDB00157 8.9E-03 5.6E-03 4.2E-03 9.7E-03 9.4E-03 5.0E-03 6.2E-03 2.4E-03 8.0E-03 2.6E-03 1.292 0.431
C_0065 Guanine C00242 HMDB00132 2.1E-05 2.1E-05 1.9E-05 2.2E-05 3.1E-05 2.3E-05 2.0E-05 1.4E-06 2.6E-05 5.0E-06 1.263 0.202
C_0045 Asp C00049, C00402, C16433 HMDB00191, HMDB06483 2.7E-03 2.7E-03 2.7E-03 3.6E-03 3.4E-03 3.4E-03 2.7E-03 2.8E-05 3.4E-03 1.2E-04 1.259 0.007 **
C_0112 Guanosine C00387 HMDB00133 1.8E-03 1.1E-03 1.0E-03 1.8E-03 1.9E-03 1.2E-03 1.3E-03 4.1E-04 1.6E-03 3.9E-04 1.233 0.405
C_0086 SDMA – HMDB03334 2.4E-05 2.4E-05 2.0E-05 2.8E-05 3.1E-05 2.4E-05 2.3E-05 2.2E-06 2.8E-05 3.7E-06 1.215 0.139
C_0058 Spermidine C00315 HMDB01257 9.9E-04 9.4E-04 1.1E-03 1.0E-03 1.5E-03 1.2E-03 1.0E-03 1.0E-04 1.2E-03 2.6E-04 1.200 0.308
C_0116 Arg-Glu – – 8.2E-06 7.6E-06 8.3E-06 8.3E-06 9.5E-06 1.1E-05 8.0E-06 3.5E-07 9.6E-06 1.3E-06 1.194 0.167
C_0061 Gln C00064, C00303, C00819 HMDB00641, HMDB03423 7.0E-03 7.0E-03 7.3E-03 8.2E-03 8.2E-03 9.0E-03 7.1E-03 1.7E-04 8.5E-03 4.6E-04 1.188 0.025 *
C_0008 1-Methyl-2-pyrrolidone C11118 – 1.2E-04 9.6E-05 1.0E-04 1.2E-04 1.1E-04 1.4E-04 1.1E-04 1.1E-05 1.2E-04 1.5E-05 1.170 0.171
C_0019 Cytosine C00380 HMDB00630 4.1E-06 3.1E-06 3.3E-06 4.2E-06 4.1E-06 3.9E-06 3.5E-06 5.4E-07 4.1E-06 1.8E-07 1.152 0.220
C_0013 2-Aminoisobutyric acid C03665 HMDB01906 9.8E-05 1.2E-04 9.4E-05 1.3E-04 1.1E-04 1.2E-04 1.0E-04 1.3E-05 1.2E-04 7.3E-06 1.141 0.177
C_0094 Carnosine C00386 HMDB00033 2.4E-03 2.4E-03 2.3E-03 3.0E-03 2.9E-03 2.2E-03 2.4E-03 7.3E-05 2.7E-03 4.6E-04 1.140 0.340
C_0021 Uracil C00106 HMDB00300 3.3E-04 2.9E-04 2.6E-04 3.8E-04 3.6E-04 2.6E-04 2.9E-04 3.9E-05 3.3E-04 6.0E-05 1.139 0.392
C_0093 Cystathionine C00542, C02291 HMDB00099 6.6E-04 4.4E-04 6.6E-04 5.7E-04 8.3E-04 6.0E-04 5.9E-04 1.2E-04 6.7E-04 1.4E-04 1.137 0.497
C_0067 His C00135, C00768, C06419 HMDB00177 5.5E-03 5.9E-03 5.4E-03 6.1E-03 6.1E-03 6.8E-03 5.6E-03 2.5E-04 6.3E-03 4.3E-04 1.135 0.071
C_0100 Homocarnosine C00884 HMDB00745 5.1E-03 4.7E-03 5.5E-03 5.4E-03 6.4E-03 5.6E-03 5.1E-03 4.3E-04 5.8E-03 5.4E-04 1.130 0.173
C_0014 Choline C00114 HMDB00097 2.3E-02 2.0E-02 1.4E-02 2.7E-02 2.3E-02 1.4E-02 1.9E-02 4.6E-03 2.1E-02 6.3E-03 1.118 0.643
C_0020 Histamine C00388 HMDB00870 2.5E-05 2.5E-05 3.0E-05 2.3E-05 4.1E-05 2.6E-05 2.7E-05 3.2E-06 3.0E-05 9.3E-06 1.116 0.630
C_0046 Adenine C00147 HMDB00034 3.6E-04 2.9E-04 2.5E-04 3.2E-04 3.9E-04 2.9E-04 3.0E-04 5.4E-05 3.3E-04 5.1E-05 1.114 0.470
C_0122 S-Adenosyl methionine C00019 HMDB01185 4.9E-04 4.4E-04 5.2E-04 5.2E-04 5.5E-04 5.4E-04 4.8E-04 4.1E-05 5.4E-04 1.8E-05 1.114 0.134
C_0030 Betaine aldehyde + H2O C00576 HMDB01252 1.7E-05 1.4E-05 1.2E-05 1.9E-05 1.5E-05 1.3E-05 1.5E-05 2.6E-06 1.6E-05 3.0E-06 1.108 0.532
C_0034 1-Methylhistamine C05127 HMDB00898 7.1E-05 4.2E-05 6.7E-05 5.6E-05 7.7E-05 6.6E-05 6.0E-05 1.6E-05 6.6E-05 1.1E-05 1.104 0.610
C_0114 Argininosuccinic acid C03406 HMDB00052 2.0E-04 1.9E-04 1.8E-04 1.9E-04 2.1E-04 2.2E-04 1.9E-04 9.3E-06 2.1E-04 1.7E-05 1.099 0.191
C_0078 Serotonin C00780 HMDB00259 4.4E-05 4.4E-05 4.4E-05 3.6E-05 6.4E-05 4.5E-05 4.4E-05 3.1E-07 4.8E-05 1.4E-05 1.099 0.649
C_0103 Uridine C00299 HMDB00296 1.5E-03 1.2E-03 1.2E-03 1.4E-03 1.6E-03 1.3E-03 1.3E-03 1.8E-04 1.4E-03 1.8E-04 1.096 0.443
C_0087 Spermine C00750 HMDB01256 7.6E-05 9.7E-05 7.7E-05 8.6E-05 7.9E-05 1.1E-04 8.3E-05 1.2E-05 9.1E-05 1.4E-05 1.091 0.524
C_0002 Gly C00037 HMDB00123 2.1E-02 1.7E-02 1.6E-02 1.9E-02 2.4E-02 1.7E-02 1.8E-02 2.3E-03 2.0E-02 3.7E-03 1.090 0.563
C_0076 Arg C00062, C00792 HMDB00517, HMDB03416 8.6E-03 6.7E-03 8.8E-03 8.3E-03 9.4E-03 8.5E-03 8.0E-03 1.2E-03 8.7E-03 6.2E-04 1.087 0.429
C_0023 Pro C00148, C00763, C16435 HMDB00162, HMDB03411 6.6E-03 6.2E-03 6.3E-03 7.5E-03 6.7E-03 6.4E-03 6.4E-03 2.3E-04 6.9E-03 5.8E-04 1.076 0.280
C_0063 Met C00073, C00855, C01733 HMDB00696 1.9E-03 1.7E-03 2.2E-03 2.1E-03 2.0E-03 2.1E-03 1.9E-03 2.2E-04 2.1E-03 4.5E-05 1.075 0.375
C_0113 His-Glu – – 3.9E-06 3.0E-06 N.D. 3.7E-06 3.5E-06 4.0E-06 3.5E-06 6.4E-07 3.7E-06 2.8E-07 1.068 0.695
C_0071 Methionine sulfoxide C02989 HMDB02005 2.5E-04 1.8E-04 2.7E-04 2.1E-04 2.9E-04 2.5E-04 2.3E-04 4.7E-05 2.5E-04 4.2E-05 1.067 0.691
C_0108 Adenosine C00212 HMDB00050 2.3E-02 2.2E-02 2.3E-02 2.5E-02 2.3E-02 2.4E-02 2.2E-02 9.2E-04 2.4E-02 5.6E-04 1.062 0.102
C_0005 β-Ala C00099 HMDB00056 2.2E-03 1.9E-03 2.1E-03 1.8E-03 2.6E-03 2.1E-03 2.1E-03 1.8E-04 2.2E-03 3.9E-04 1.062 0.650
C_0105 γ-Glu-Cys C00669 HMDB01049 1.3E-04 1.1E-04 1.3E-04 1.6E-04 1.2E-04 1.2E-04 1.2E-04 1.5E-05 1.3E-04 2.1E-05 1.060 0.650
C_0110 Saccharopine C00449 HMDB00279 3.8E-04 3.5E-04 3.7E-04 2.9E-04 4.9E-04 3.7E-04 3.7E-04 1.7E-05 3.9E-04 1.0E-04 1.058 0.754
C_0082 N6-Acetyllysine C02727 HMDB00206 2.8E-05 2.3E-05 3.0E-05 2.9E-05 3.0E-05 2.7E-05 2.7E-05 3.9E-06 2.9E-05 1.8E-06 1.053 0.605
C_0015 GABA C00334 HMDB00112 5.7E-03 4.6E-03 4.9E-03 4.4E-03 6.2E-03 5.3E-03 5.1E-03 5.9E-04 5.3E-03 9.1E-04 1.048 0.722
C_0068 2-Aminoadipic acid C00956 HMDB00510 1.5E-03 1.9E-03 2.0E-03 1.8E-03 1.7E-03 2.2E-03 1.8E-03 2.7E-04 1.9E-03 2.9E-04 1.048 0.726
C_0040 Asn C00152, C01905, C16438 HMDB00168 3.4E-03 3.5E-03 3.7E-03 3.8E-03 3.6E-03 3.6E-03 3.5E-03 1.7E-04 3.7E-03 1.3E-04 1.046 0.275
C_0097 Thr-Asp – – 1.2E-05 1.4E-05 1.1E-05 1.1E-05 1.4E-05 1.4E-05 1.2E-05 1.5E-06 1.3E-05 2.0E-06 1.045 0.721
C_0075 N-Acetylornithine C00437 HMDB03357 1.3E-05 8.3E-06 ND 1.1E-05 ND ND 1.1E-05 3.5E-06 1.1E-05 NA 1.043 NA
C_0025 Betaine C00719 HMDB00043 9.6E-04 8.3E-04 9.5E-04 9.2E-04 1.1E-03 8.3E-04 9.1E-04 7.0E-05 9.5E-04 1.4E-04 1.042 0.696
C_0010 Homoserine lactone – – 7.8E-05 6.8E-05 8.0E-05 7.3E-05 7.7E-05 8.4E-05 7.5E-05 6.9E-06 7.8E-05 5.6E-06 1.032 0.660
C_0107 Thiamine C00378 HMDB00235 6.8E-05 6.0E-05 5.3E-05 6.7E-05 6.2E-05 5.8E-05 6.0E-05 7.7E-06 6.2E-05 4.7E-06 1.029 0.753
C_0111 1-Methyladenosine C02494 HMDB03331 9.6E-05 1.1E-04 1.5E-04 1.2E-04 1.2E-04 1.2E-04 1.2E-04 2.6E-05 1.2E-04 3.8E-06 1.028 0.850
C_0064 Triethanolamine C06771 – 1.1E-05 1.2E-05 1.4E-05 1.5E-05 1.2E-05 1.2E-05 1.2E-05 1.5E-06 1.3E-05 1.6E-06 1.028 0.799
C_0026 Val C00183, C06417, C16436 HMDB00883 8.5E-03 7.5E-03 7.5E-03 7.8E-03 8.2E-03 8.0E-03 7.8E-03 5.5E-04 8.0E-03 1.9E-04 1.022 0.653
C_0056 Acetylcholine C01996 HMDB00895 4.5E-04 5.0E-04 6.0E-04 4.8E-04 4.9E-04 6.1E-04 5.2E-04 7.8E-05 5.3E-04 7.3E-05 1.021 0.869
C_0115 5′-Deoxy-5′-methyl thioadenosine C00170 HMDB01173 1.9E-05 1.6E-05 1.7E-05 2.1E-05 1.6E-05 1.6E-05 1.8E-05 1.3E-06 1.8E-05 2.9E-06 1.016 0.891
C_0054 Stachydrine C10172 HMDB04827 1.3E-04 9.7E-05 1.4E-04 1.5E-04 1.3E-04 8.6E-05 1.2E-04 2.2E-05 1.2E-04 3.4E-05 1.014 0.946
C_0085 Gly-Asp - - 8.2E-05 9.0E-05 8.5E-05 1.0E-04 8.4E-05 7.3E-05 8.6E-05 4.3E-06 8.6E-05 1.4E-05 1.009 0.935
C_0039 Leu C00123, C01570, C16439 HMDB00687 9.5E-03 8.3E-03 8.4E-03 8.9E-03 9.0E-03 8.4E-03 8.7E-03 7.0E-04 8.8E-03 3.1E-04 1.004 0.941
C_0080 Phosphorylcholine C00588 HMDB01565 1.6E-02 1.6E-02 1.8E-02 1.6E-02 1.6E-02 1.6E-02 1.6E-02 1.1E-03 1.6E-02 2.3E-04 0.994 0.891
C_0036 trans-Glutaconic acid C02214 HMDB00620 9.0E-05 9.7E-05 9.9E-05 7.7E-05 1.1E-04 9.7E-05 9.5E-05 4.7E-06 9.4E-05 1.7E-05 0.992 0.945
C_0090 Carboxymethyl lysine – – 5.5E-05 5.2E-05 5.2E-05 5.1E-05 5.7E-05 5.0E-05 5.3E-05 2.1E-06 5.2E-05 3.7E-06 0.989 0.818
C_0095 2′-Deoxycytidine C00881 HMDB00014 3.3E-05 2.2E-05 2.6E-05 3.5E-05 2.7E-05 1.9E-05 2.7E-05 5.6E-06 2.7E-05 7.8E-06 0.988 0.955
C_0038 Ile C00407, C06418, C16434 HMDB00172 4.8E-03 4.2E-03 4.1E-03 4.4E-03 4.3E-03 4.3E-03 4.4E-03 4.1E-04 4.3E-03 6.2E-05 0.987 0.841
C_0012 2-Aminobutyric acid C02261, C02356 HMDB00452 1.7E-04 1.4E-04 1.7E-04 1.7E-04 1.5E-04 1.6E-04 1.6E-04 2.0E-05 1.6E-04 7.1E-06 0.987 0.879
C_0062 Glu C00025, C00217, C00302 HMDB00148, HMDB03339 1.4E-02 1.4E-02 1.5E-02 1.4E-02 1.3E-02 1.5E-02 1.4E-02 4.6E-04 1.4E-02 6.3E-04 0.987 0.698
C_0022 Creatinine C00791 HMDB00562 6.6E-04 5.9E-04 6.5E-04 5.6E-04 6.3E-04 6.7E-04 6.3E-04 4.0E-05 6.2E-04 5.5E-05 0.978 0.745
C_0083 Gly-Leu C02155 HMDB00759 1.5E-04 1.4E-04 1.5E-04 1.6E-04 1.4E-04 1.3E-04 1.5E-04 9.7E-06 1.4E-04 1.4E-05 0.978 0.759
C_0059 Lys C00047, C00739, C16440 HMDB00182, HMDB03405 1.1E-02 9.5E-03 1.0E-02 9.8E-03 1.1E-02 1.0E-02 1.0E-02 8.8E-04 1.0E-02 4.4E-04 0.977 0.702
C_0084 N6,N6,N6-Trimethyllysine C03793 HMDB01325 3.4E-04 4.3E-04 4.1E-04 4.0E-04 3.3E-04 4.2E-04 3.9E-04 4.7E-05 3.8E-04 4.7E-05 0.977 0.823
C_0104 Pyridoxamine 5′-phosphate C00647 HMDB01555 3.4E-04 3.5E-04 3.7E-04 3.2E-04 3.7E-04 3.5E-04 3.5E-04 1.4E-05 3.5E-04 2.4E-05 0.976 0.641
C_0117 Glutathione (GSSG)_divalent C00127 HMDB03337 1.8E-02 2.0E-02 1.7E-02 1.7E-02 2.1E-02 1.6E-02 1.9E-02 1.4E-03 1.8E-02 2.7E-03 0.973 0.794
C_0092 N-Acetylglucosamine C00140 HMDB00215 1.5E-04 1.6E-04 1.4E-04 1.6E-04 1.5E-04 1.3E-04 1.5E-04 6.8E-06 1.5E-04 1.3E-05 0.971 0.639
C_0069 Carnitine C00318, C00487, C15025 HMDB00062 8.6E-03 7.7E-03 7.9E-03 7.4E-03 9.0E-03 7.1E-03 8.1E-03 4.9E-04 7.8E-03 1.0E-03 0.969 0.736
C_0001 Urea C00086 HMDB00294 7.5E-02 7.4E-02 7.7E-02 7.0E-02 7.3E-02 7.5E-02 7.5E-02 1.6E-03 7.3E-02 2.7E-03 0.968 0.274
C_0042 Creatine C00300 HMDB00064 2.2E-02 2.2E-02 2.3E-02 2.1E-02 2.1E-02 2.2E-02 2.2E-02 4.4E-04 2.1E-02 3.2E-04 0.965 0.078
C_0051 Tyramine C00483 HMDB00306 1.2E-05 1.0E-05 2.0E-05 1.1E-05 1.5E-05 1.3E-05 1.4E-05 5.0E-06 1.3E-05 2.0E-06 0.962 0.878
C_0007 Ala C00041, C00133, C01401 HMDB00161, HMDB01310 1.1E-03 1.2E-03 1.2E-03 1.3E-03 9.6E-04 1.2E-03 1.2E-03 4.3E-05 1.1E-03 1.6E-04 0.960 0.667
C_0077 Citrulline C00327 HMDB00904 1.1E-03 1.0E-03 1.1E-03 1.2E-03 8.2E-04 1.1E-03 1.1E-03 6.9E-05 1.0E-03 1.9E-04 0.959 0.735
C_0004 Putrescine C00134 HMDB01414 1.2E-04 9.7E-05 1.0E-04 9.5E-05 1.0E-04 1.0E-04 1.1E-04 1.2E-05 1.0E-04 4.7E-06 0.951 0.544
C_0106 Glycero phosphocholine C00670 HMDB00086 1.8E-02 1.9E-02 2.1E-02 1.3E-02 2.1E-02 2.0E-02 1.9E-02 1.5E-03 1.8E-02 4.2E-03 0.947 0.725
C_0027 Homoserine C00263 HMDB00719 2.3E-04 2.6E-04 2.7E-04 2.6E-04 2.2E-04 2.4E-04 2.5E-04 2.0E-05 2.4E-04 1.8E-05 0.940 0.379
C_0055 4-Guanidinobutyric acid C01035 HMDB03464 3.3E-04 2.6E-04 3.3E-04 3.0E-04 3.0E-04 2.5E-04 3.1E-04 4.4E-05 2.8E-04 3.3E-05 0.929 0.534
C_0119 Thiamine phosphate C01081 HMDB02666 5.2E-05 4.8E-05 4.1E-05 4.3E-05 4.8E-05 3.9E-05 4.7E-05 5.6E-06 4.4E-05 4.4E-06 0.924 0.436
C_0043 Ornithine C00077, C00515, C01602 HMDB00214, HMDB03374 3.4E-04 2.7E-04 4.3E-04 3.5E-04 2.8E-04 3.2E-04 3.5E-04 7.7E-05 3.2E-04 3.5E-05 0.917 0.597
C_0102 Cytidine C00475 HMDB00089 2.0E-03 1.8E-03 1.6E-03 2.1E-03 1.4E-03 1.4E-03 1.8E-03 2.2E-04 1.6E-03 4.1E-04 0.891 0.520
C_0033 Taurine C00245 HMDB00251 2.8E-02 3.0E-02 3.2E-02 2.8E-02 2.4E-02 2.7E-02 3.0E-02 1.8E-03 2.7E-02 2.5E-03 0.885 0.128
C_0028 Thr C00188, C00820 HMDB00167 1.8E-02 1.8E-02 1.9E-02 1.4E-02 1.6E-02 1.7E-02 1.8E-02 4.3E-04 1.6E-02 1.3E-03 0.884 0.101
C_0118 Glutathione (GSH) C00051 HMDB00125 5.6E-03 6.1E-03 7.3E-03 5.5E-03 4.2E-03 6.7E-03 6.3E-03 8.6E-04 5.5E-03 1.2E-03 0.874 0.422
C_0121 S-Adenosylhomo cysteine C00021 HMDB00939 6.8E-05 7.6E-05 7.7E-05 6.7E-05 6.7E-05 5.9E-05 7.4E-05 5.0E-06 6.4E-05 4.8E-06 0.874 0.080
C_0091 β-Ala-Lys C05341 – 3.4E-05 2.4E-05 3.8E-05 2.4E-05 3.6E-05 2.4E-05 3.2E-05 7.1E-06 2.8E-05 7.0E-06 0.873 0.521
C_0024 Guanidoacetic acid C00581 HMDB00128 4.6E-04 3.8E-04 4.2E-04 4.0E-04 3.4E-04 3.3E-04 4.2E-04 4.1E-05 3.6E-04 3.8E-05 0.855 0.131
C_0057 γ-Butyrobetaine C01181 HMDB01161 1.8E-03 1.7E-03 1.8E-03 1.6E-03 1.3E-03 1.5E-03 1.8E-03 3.1E-05 1.5E-03 1.3E-04 0.841 0.059
C_0017 N-Methylaniline C02299 – ND 1.6E-05 1.5E-05 1.1E-05 1.4E-05 ND 1.5E-05 9.7E-07 1.3E-05 2.2E-06 0.834 0.325
C_0070 5-Hydroxylysine C16741 HMDB00450 1.4E-05 1.0E-05 1.2E-05 1.1E-05 7.4E-06 1.1E-05 1.2E-05 1.8E-06 1.0E-05 2.3E-06 0.831 0.295
C_0096 γ-Glu-2-aminobutyric acid – – 1.7E-04 1.6E-04 2.0E-04 1.5E-04 1.6E-04 1.3E-04 1.8E-04 1.9E-05 1.5E-04 1.3E-05 0.829 0.090
C_0088 O-Acetylcarnitine C02571 HMDB00201 5.0E-04 2.9E-04 4.6E-04 3.8E-04 3.3E-04 3.1E-04 4.2E-04 1.1E-04 3.4E-04 3.4E-05 0.816 0.370
C_0041 Gly-Gly C02037 HMDB11733 1.2E-04 1.4E-04 1.5E-04 1.1E-04 1.0E-04 1.2E-04 1.4E-04 1.3E-05 1.1E-04 1.1E-05 0.804 0.053
C_0101 5,6,7,8-Tetrahydrobiopterin C00272 HMDB00027 N.D. 1.3E-05 1.5E-05 7.9E-06 1.2E-05 1.4E-05 1.4E-05 1.6E-06 1.1E-05 3.1E-06 0.794 0.264
C_0016 Ser C00065, C00716, C00740 HMDB00187, HMDB03406 9.8E-04 1.1E-03 1.1E-03 8.2E-04 7.3E-04 8.3E-04 1.0E-03 6.5E-05 7.9E-04 5.5E-05 0.759 0.007 **
C_0081 N8-Acetylspermidine C01029 HMDB02189 5.6E-05 4.6E-05 4.6E-05 3.9E-05 3.5E-05 3.6E-05 4.9E-05 6.0E-06 3.7E-05 2.0E-06 0.746 0.056
C_0050 Trigonelline C01004 HMDB00875 1.4E-04 9.8E-05 1.2E-04 1.0E-04 8.2E-05 7.4E-05 1.2E-04 1.8E-05 8.7E-05 1.6E-05 0.738 0.095
C_0048 1-Methylnicotinamide C02918 HMDB00699 4.5E-05 5.3E-05 5.4E-05 4.3E-05 3.4E-05 3.4E-05 5.0E-05 5.2E-06 3.7E-05 5.0E-06 0.736 0.033 *
C_0098 Ser-Glu – – 5.0E-05 4.1E-05 5.0E-05 3.5E-05 3.8E-05 2.9E-05 4.7E-05 5.5E-06 3.4E-05 4.8E-06 0.729 0.040 *
C_0052 Urocanic acid C00785 HMDB00301 1.0E-04 3.6E-05 4.4E-05 4.1E-05 6.9E-05 1.2E-05 6.0E-05 3.5E-05 4.1E-05 2.8E-05 0.677 0.503
C_0037 Hydroxyproline C01015, C01157 HMDB06055, HMDB00725 1.6E-03 1.7E-03 1.6E-03 1.2E-03 9.9E-04 1.0E-03 1.6E-03 3.2E-05 1.1E-03 1.1E-04 0.651 0.009 **
C_0009 Cyclohexylamine C00571 – 1.5E-05 1.9E-05 2.3E-05 1.4E-05 8.5E-06 1.4E-05 1.9E-05 4.1E-06 1.2E-05 3.4E-06 0.647 0.094
C_0120 S-Lactoylglutathione C03451 HMDB01066 ND ND 1.8E-05 ND ND 1.0E-05 1.8E-05 NA 1.0E-05 NA 0.584 NA
C_0066 Dopamine C03758 HMDB00073 2.9E-04 5.8E-04 7.1E-04 2.4E-04 2.7E-04 3.5E-04 5.3E-04 2.2E-04 2.9E-04 5.6E-05 0.543 0.188
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1Ex-GF/GF ratio.

2Welch’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001, NA, not available).

ND, not detected.

Abbreviations

BBB, blood-brain barrier; CE-TOFMS, capillary electrophoresis with time-of-flight mass spectrometry; CNS, central nervous system; DA, dopamine; Ex-GF, ex-germ-free; GF, germ-free; MGB axis, microbiota-gut-brain axis; RSD, relative standard deviations.

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