# Deciphering the Metabolomics-Based Intervention of Yanghe Decoction on Hashimoto’s Thyroiditis

Authors: Xing Zhang; Dexuan Chen; Kun Xu; Zhaoqun Ma
Journal: Evidence-Based Complementary and Alternative Medicine
 (2022)

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## Abstract

Background. Yanghe decoction is a famous formula consisting of Rehmannia, deer horn gum, cinnamon, rue, Ephedra, ginger charcoal, and licorice. However, few studies have explored the role of the potential mechanism of Yanghe decoction in the treatment of Hashimoto’s thyroiditis by metabolomics. Methods. Nine mice were randomly divided into three groups: control group (group C), model group (group M), and drug administration group (group T), with three mice in each group. Mice in groups M and T were established as models of Hashimoto’s thyroiditis, and group T was treated with Yanghe decoction. The metabolome of plasma samples from each group of mice was determined using mass spectrometry coupled with high-performance liquid and gas phases, and nuclear magnetic resonance. Based on the three assays, principal component analysis was performed on all samples, as well as orthogonal partial least squares-discriminant analysis and differential metabolite molecules for groups M and T. Subsequently, pathway enrichment analysis was performed, and the intersection was taken for the differential metabolites screened in the M and T groups. The levels of inflammatory factors IL-35 and IL-6 within the serum of each group of mice were detected. Results. The difference analysis showed that a total of 38 differential metabolites were screened based on mass spectrometry coupled with the high-performance liquid phase, 120 differential metabolites were screened based on mass spectrometry coupled with gas phase, and a total of α-glucose and β-glucose were the differential metabolites analyzed based on NMR test results. The pathways enriched by the differential metabolites in the M and T groups were intersected, and a total of 5 common pathways were obtained (amino acid tRNA biosynthesis, D-glutamine and D-glutamate metabolism, tryptophan metabolism, nitrogen metabolism, and arginine and proline metabolism). The results also showed a significant decrease in the serum inflammatory factor IL-35 and a significant increase in IL-6 in mice from group M compared with group C, while a significant increase in the serum inflammatory factor IL-35 and a significant decrease in IL-6 in mice from group T compared with group M. Conclusion. Our study reveals the metabolites as well as a metabolic network that can be altered by Yanghe decoction treatment of Hashimoto’s thyroiditis and shows that Yanghe decoction can effectively reduce the level of inflammatory factors in Hashimoto’s thyroid.


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## Body

## 1. Introduction

Hashimoto’s thyroiditis, also known as chronic lymphocytic or autoimmune thyroiditis, is an autoimmune thyroid disease that causes the immune system to attack and destroy the thyroid gland [1]. It is characterized by an enlarged thyroid gland, parenchymal lymphocytic infiltration, and the presence of thyroid antigen-specific antibodies [2]. Hashimoto’s thyroiditis causes chronic inflammation of the thyroid tissue and may result in hypothyroidism in 20–30% of patients [3]. The incidence is approximately 3 to 6 cases per 10,000 people per year, with a prevalence of at least 2% in women. The glands involved in thyroiditis often lose the ability to store iodine, produce and secrete rod proteins that circulate in the plasma, and fail to make hormones efficiently [4,5]. At present, methylprednisolone is often used in clinical practice for Hashimoto’s thyroiditis treatment, which could inhibit the synthesis of the thyroid gland and alleviate the patient’s clinical symptoms. However, research has shown that the use of methimazole alone in patients with Hashimoto’s thyroiditis for long term often have poor prognosis.Traditional Chinese medicine is remarkably effective in the adjuvant treatment of the disease, especially in improving clinical symptoms, prolonging patient survival, and modulating immune function. As an effective method for preventing and treating diseases, TCM has been increasingly used worldwide in the past decades [6]. Yanghe decoction is a famous formula consisting of Rehmannia, deer horn gum, cinnamon, rue, Ephedra, ginger charcoal, and licorice [7]. It has the effect of warming yang and nourishing blood and dispersing cold and moving stagnation. For centuries, Yang He Tang has been proven to be used to treat a variety of noninfectious inflammatory conditions [8].Yanghe decoction can be combined with modern therapies, which have a combination of “multicomponent, multitarget, and multipathway” regulatory mechanisms and have fewer toxic side effects [9]. In one study, Yang He Tang was shown to have a high mean cure rate (defined as complete regression or significant improvement of lumps and pain for at least two months) in patients with chronic breast fibrosis and palpable lumps [10]. Fewer studies have clinically investigated the effect of Yanghe decoction in the treatment of Hashimoto’s thyroiditis. Therefore, the present study was conducted to investigate the potential mechanism of Yanghe decoction in the treatment of Hashimoto’s thyroiditis through a metabolomic approach.


## 2. Material and Methods

### 2.1. Animal Grouping and Model Construction

Nine NOD mice (4 weeks old) were purchased from the Nanjing Biomedical Research Institute of Nanjing University. NOD mice were randomly divided into three groups: control group (group C), model group (group M), and drug administration group (group T), with three mice in each group. The mice in the model group and the drug administration group were treated with porcine thyroglobulin and high iodine water to establish the mouse model of autoimmune thyroiditis. After successful modeling, Yanghe decoction formula granule (Xuyang Pharmaceutical Co., LTD., China) was prepared into 0.5 g/mL liquid medicine; the drug-administered group was given 0.5 g/ml Yanghe decoction formula 1 ml/100 g by gavage for 1 h, and the model group and normal group were given the same amount of saline by gavage once a day for 10 weeks. The serum and plasma samples were collected and stored at −20°C for serum and −80°C for plasma. The study protocols were approved by the Institutional Animal Care and Use Committee of Nanjing University of Chinese Medicine Affiliated Yancheng.


### 2.2. Enzyme-Linked Immunosorbent Assay

The serum collected was assayed for IL-35 (interleukin-35) and IL-6 (interleukin-6) inflammatory factors using enzyme-linked immunosorbent assay (ELISA) kits. The experiment was performed according to the instructions.


### 2.3. Mass Spectrometry Coupled with Ultra-High Performance Liquid-Phase Detection

Metabolite extraction was performed in strict accordance with the operating instructions, followed by onboard detection.On-Board Detection. The target compounds were chromatographed on a Waters ACQUITY UPLCBEH Amide (2.1 mm × 100 mm, 1.7 μm) column using a Vanquish (Thermo Fisher Scientific) ultra-performance liquid chromatography. The A phase of the liquid chromatography was aqueous containing 25 mmol/L ammonium acetate and 25 mmol/L ammonia, and the B phase was acetonitrile. The sample tray temperature is 4°C, and injection volume is 2 μL.The Thermo Q Exactive HFX mass spectrometer was capable of primary and secondary mass spectrometry data acquisition under the control of the control software Xcalibur (Thermo). Detailed parameters were as follows: sheath gas flow rate, 30 Arb; Aux gas flow rate, 25 Arb; capillary temperature: 350°C; full ms resolution, 60000; MS/MS resolution, 7500; collision energy, 10/30/60 in NCE mode; and spray voltage: 3.6 kV (positive) or −3.2 kV (negative).Data Processing. The raw data were converted to mzXML format by ProteoWizard software, and then the peak identification, peak extraction, peak alignment, and integration were performed using the R package (kernel XCMS) written by ourselves and then matched with the BiotreeDB (V2.1) self-built secondary mass spectrometry database for substance annotation.


### 2.4. Mass Spectrometry Coupled with Gas Chromatography Detection

Metabolite extraction was performed strictly according to the operating instructions, and then all samples were analyzed by gas chromatography and time-of-flight mass spectrometry.On-Board Detection. GC-TOF-MS analysis was performed using an Agilent 7890 gas chromatograph and a time-of-flight mass spectrometer. The system used a DB-5MS capillary column. 1 μL aliquots were injected in a nonsplit mode. Helium was used as the carrier gas with a front inlet purge flow rate of 3 mL min−1 and a gas flow rate through the column of 1 ml min−1. The initial temperature was held at 50°C for 1 min, then increased to 310°C at a rate of 20°C, and then held at 310°C for 6 min. The injection, transmission line, and ion source temperatures were 280, 280, and 250°C, respectively. The energy in electron collision mode was −70 eV. After a solvent delay of 4.83 min, mass spectral data were acquired in full-scan mode in the m/z range of 50–500 at a rate of 12.5 spectra per second.Data Processing. The mass spectral data were analyzed for peak extraction, baseline correction, deconvolution, peak integration, and peak alignment using ChromaTOF software (V 4.3x, LECO). For the substance characterization work, the LECO-Fiehn Rtx5 database was used, including mass spectrometry matching and retention time index matching.


### 2.5. Nuclear Magnetic Resonance Detection

The experimental testing equipment was Varian Inova 600M Agilent.Description of the Spectral Processing. The integration interval of the serum NMR spectra was 0.5–8.5 ppm with an integration spacing of 0.002 ppm, and a section of 4.60–4.80 ppm containing the residual water peak and the urea peak at 5.20–5.25 ppm and the region of its influence was removed. Among them, the demonstration of mouse serum spectra for groups C, M, and T were chosen: 1, 9, and 17, respectively.


### 2.6. Principal Component Analysis (PCA)

Data were formatted for logarithmic (LOG) transformation plus centralization (CTR) using SIMCA software (V16.0.2; Sartorius Stedim Data Analytics AB, Umea, Sweden), followed by automated modeling analysis.


### 2.7. Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA)

The data were UV-formatted using SIMCA software (V14.1; MKS Data Analytics Solutions, Umea, Sweden), and OPLS-DA modeling analysis was first performed on the first principal component, and the quality of the model was tested with 7-fold cross-validation (7-fold cross-validation). Then, R2Y (interpretability of the model for the categorical variable Y) and Q2 (predictability of the model) were used to judge the validity of the model; finally, the validity of the model was further tested by a permutation test, which randomly changed the order of the categorical variable Y several times to obtain different random Q2 values.


### 2.8. Pathway Enrichment Analysis

The metabolic pathways of differentially expressed metabolites were analyzed by searching the relevant metabolic pathways of differentially expressed metabolites using authoritative metabolite databases such as Kyoto Encyclopedia of Genes and Genomes (KEGG) and PubChem.


## 2.1. Animal Grouping and Model Construction

Nine NOD mice (4 weeks old) were purchased from the Nanjing Biomedical Research Institute of Nanjing University. NOD mice were randomly divided into three groups: control group (group C), model group (group M), and drug administration group (group T), with three mice in each group. The mice in the model group and the drug administration group were treated with porcine thyroglobulin and high iodine water to establish the mouse model of autoimmune thyroiditis. After successful modeling, Yanghe decoction formula granule (Xuyang Pharmaceutical Co., LTD., China) was prepared into 0.5 g/mL liquid medicine; the drug-administered group was given 0.5 g/ml Yanghe decoction formula 1 ml/100 g by gavage for 1 h, and the model group and normal group were given the same amount of saline by gavage once a day for 10 weeks. The serum and plasma samples were collected and stored at −20°C for serum and −80°C for plasma. The study protocols were approved by the Institutional Animal Care and Use Committee of Nanjing University of Chinese Medicine Affiliated Yancheng.


## 2.2. Enzyme-Linked Immunosorbent Assay

The serum collected was assayed for IL-35 (interleukin-35) and IL-6 (interleukin-6) inflammatory factors using enzyme-linked immunosorbent assay (ELISA) kits. The experiment was performed according to the instructions.


## 2.3. Mass Spectrometry Coupled with Ultra-High Performance Liquid-Phase Detection

Metabolite extraction was performed in strict accordance with the operating instructions, followed by onboard detection.On-Board Detection. The target compounds were chromatographed on a Waters ACQUITY UPLCBEH Amide (2.1 mm × 100 mm, 1.7 μm) column using a Vanquish (Thermo Fisher Scientific) ultra-performance liquid chromatography. The A phase of the liquid chromatography was aqueous containing 25 mmol/L ammonium acetate and 25 mmol/L ammonia, and the B phase was acetonitrile. The sample tray temperature is 4°C, and injection volume is 2 μL.The Thermo Q Exactive HFX mass spectrometer was capable of primary and secondary mass spectrometry data acquisition under the control of the control software Xcalibur (Thermo). Detailed parameters were as follows: sheath gas flow rate, 30 Arb; Aux gas flow rate, 25 Arb; capillary temperature: 350°C; full ms resolution, 60000; MS/MS resolution, 7500; collision energy, 10/30/60 in NCE mode; and spray voltage: 3.6 kV (positive) or −3.2 kV (negative).Data Processing. The raw data were converted to mzXML format by ProteoWizard software, and then the peak identification, peak extraction, peak alignment, and integration were performed using the R package (kernel XCMS) written by ourselves and then matched with the BiotreeDB (V2.1) self-built secondary mass spectrometry database for substance annotation.


## 2.4. Mass Spectrometry Coupled with Gas Chromatography Detection

Metabolite extraction was performed strictly according to the operating instructions, and then all samples were analyzed by gas chromatography and time-of-flight mass spectrometry.On-Board Detection. GC-TOF-MS analysis was performed using an Agilent 7890 gas chromatograph and a time-of-flight mass spectrometer. The system used a DB-5MS capillary column. 1 μL aliquots were injected in a nonsplit mode. Helium was used as the carrier gas with a front inlet purge flow rate of 3 mL min−1 and a gas flow rate through the column of 1 ml min−1. The initial temperature was held at 50°C for 1 min, then increased to 310°C at a rate of 20°C, and then held at 310°C for 6 min. The injection, transmission line, and ion source temperatures were 280, 280, and 250°C, respectively. The energy in electron collision mode was −70 eV. After a solvent delay of 4.83 min, mass spectral data were acquired in full-scan mode in the m/z range of 50–500 at a rate of 12.5 spectra per second.Data Processing. The mass spectral data were analyzed for peak extraction, baseline correction, deconvolution, peak integration, and peak alignment using ChromaTOF software (V 4.3x, LECO). For the substance characterization work, the LECO-Fiehn Rtx5 database was used, including mass spectrometry matching and retention time index matching.


## 2.5. Nuclear Magnetic Resonance Detection

The experimental testing equipment was Varian Inova 600M Agilent.Description of the Spectral Processing. The integration interval of the serum NMR spectra was 0.5–8.5 ppm with an integration spacing of 0.002 ppm, and a section of 4.60–4.80 ppm containing the residual water peak and the urea peak at 5.20–5.25 ppm and the region of its influence was removed. Among them, the demonstration of mouse serum spectra for groups C, M, and T were chosen: 1, 9, and 17, respectively.


## 2.6. Principal Component Analysis (PCA)

Data were formatted for logarithmic (LOG) transformation plus centralization (CTR) using SIMCA software (V16.0.2; Sartorius Stedim Data Analytics AB, Umea, Sweden), followed by automated modeling analysis.


## 2.7. Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA)

The data were UV-formatted using SIMCA software (V14.1; MKS Data Analytics Solutions, Umea, Sweden), and OPLS-DA modeling analysis was first performed on the first principal component, and the quality of the model was tested with 7-fold cross-validation (7-fold cross-validation). Then, R2Y (interpretability of the model for the categorical variable Y) and Q2 (predictability of the model) were used to judge the validity of the model; finally, the validity of the model was further tested by a permutation test, which randomly changed the order of the categorical variable Y several times to obtain different random Q2 values.


## 2.8. Pathway Enrichment Analysis

The metabolic pathways of differentially expressed metabolites were analyzed by searching the relevant metabolic pathways of differentially expressed metabolites using authoritative metabolite databases such as Kyoto Encyclopedia of Genes and Genomes (KEGG) and PubChem.


## 3. Results

### 3.1. Principal Component Analysis

First, we performed PCA on the results of the three assays to observe the overall metabolic levels and species differences between the groups. The results of principal component analysis (positive ions) of the mass spectrometry coupled with gas and high-performance liquid phases showed that the samples of groups M and T were more closely distributed and both groups were distant from the samples of group C. The results of the NMR principal component analysis showed that the samples of groups M and T were distributed more into and partitioned from the samples of group C. This can all indicate that the more similar the type and content of metabolites in groups M and T, the more different the overall metabolic levels in groups M and T from group C. The samples were all in the 95% confidence interval. See Figure1.Figure 1
Principal component analysis. (a) Scatter plot and 3D plot of PCA scores for all samples detected by mass spectrometry coupled with the high-performance liquid phase. (b) Scatter plot and 3D plot of PCA scores for all samples detected by mass spectrometry coupled with the gas phase. (c) Scatter plot and 3D plot of PCA scores of all samples detected by NMR.
(a)(b)(c)


### 3.2. Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) and Differential Metabolite Analysis

We then modeled OPLS-DA for the M and T groups and screened for differential metabolites. The scatter plots of OPLS-DA model scores for the three assays showed that the horizontal distance between samples was farther for group M versus group T, which could indicate the greater the difference between the two groups, and the samples were very clearly differentiated. The samples were all in the 95% confidence interval. The results of the differential metabolite analysis showed that a total of 38 differential metabolites were screened for the mass spectrometry coupled with high-performance liquid phase (Table1); a total of 120 differential metabolites were screened for the mass spectrometry coupled with gas phase (Table 2), and the conditions for this metabolite screening were VIP value (the projected importance of the variable obtained from the OPLS-DA model for the comparison of the substance in this group) > 1 and P<0.05. NMR analysis showed that α-glucose and β-glucose were significant differentiating metabolites (see Figure 2).Table 1
Differential metabolites detected by mass spectrometry and high-performance liquid chromatography.
IDMS2 nameMS2 scorertmzSuperClassTypeMEAN TMEAN MVIPP-valueQ-valueFOLDCHANGELOG_FOLDCHANGE22D-Glutamine0.999291389.5485147.0759376Organic acids and derivativesForward3.7461839523.4466533471.9316642810.01476564411.0869047670.12022553947Trigonelline0.995484301.0875138.0543287Alkaloids and derivativesForward1.0249576010.8389521332.0221650480.04605110311.2217116580.28890382854N-Acetylhistamine0.994856147.787154.0968957Organic acids and derivativesForward0.0739908140.1106984541.8291806350.02558547810.668399709−0.5812169956Kanzonol K0.99465635.0689437.1927582Phenylpropanoids and polyketidesForward0.0519548910.0310480152.1012812750.00197428911.6733723930.74275853969L-Methionine0.991602301.415150.0577251Organic acids and derivativesForward0.6169196450.4680697971.9399106810.01967814411.3180078030.39835891299Ecgonine0.981134350.546186.1120848Alkaloids and derivativesForward0.0273475090.0306545131.9266898810.03169306910.892120183−0.164690017101L-histidine0.979726408.5865156.0761942Organic acids and derivativesForward0.494053370.2238941392.081970460.00858243312.2066382441.141850134107L-phenylalanine0.977905275.944166.0854692Organic acids and derivativesForward4.7637714663.8284602411.8972162090.03432289211.2443048030.315339929112L-alanine0.975801363.87990.05507238Organic acids and derivativesForward0.4379204690.3634103631.9121314980.03199470711.2050302190.269069326138Phytosphingosine0.96538157.8408318.299383Organic nitrogen compoundsforward1.7435292661.1264069121.9483855330.02199269611.5478680470.630282489141Pentanenitrile0.962912266.60484.08082795Organonitrogen compoundsForward0.2645201370.2265185882.0058009640.01882448611.1677634870.223748107153Imidazoleacetic acid riboside0.954489370.1495259.0919389Nucleosides, nucleotides, and analoguesForward0.0149655020.0194694762.0402879720.00604459310.768664826−0.3795734421563beta,6beta-Dihydroxynortropane0.94801152.1612144.1012842Alkaloids and derivativesForward0.1188181450.0864957282.0504821120.00443131111.3736880180.458054387158L-kynurenine0.944886280.281209.0914398Organic oxygen compoundsForward0.103724740.0828028411.9877219230.01317307711.2526712630.32500786160Arecaidine0.944798282.167142.0858524Alkaloids and derivativesForward0.0800852940.0530141312.0167966560.02200487211.5106405180.595160389161Carnosine0.942805434.3945227.113435Organic acids and derivativesForward0.0480449360.0663721361.8190562390.03111277110.723872074−0.466193334169Thelephoric acid0.935613407.237353.0278188Organoheterocyclic compoundsForward0.0026053810.0037754051.8061407080.03321201710.690093214−0.535136849180L-acetylcarnitine0.92253320.788204.1225337Lipids and lipid-like moleculesForward3.6985298422.8399080031.8771470180.02489894611.3023414270.381107721204Alpha-methylstyrene0.89864235.2889119.0850948BenzenoidsForward0.1884414730.1316320932.1598874960.00157772911.4315769780.517605248228Glycerophosphocholine0.86945400.206258.1089005Lipids and lipid-like moleculesForward0.6159519630.9257415111.8646435320.03582193310.665360638−0.5877915732301-(beta-D-ribofuranosyl)-1,4-dihydronicotinamide0.865109171.668257.1128126Organic oxygen compoundsForward0.1173256080.1385665812.1380359110.00478950910.846709269−0.240061413238D-biopterin0.853104272.631238.0928999Organoheterocyclic compoundsForward0.0264860360.0214462491.828256740.04043988811.2349962320.30450664240N-(3-methylbutyl)acetamide0.850798268.9475130.1221116Organic acids and derivativesForward0.0582095810.0497433652.1420982630.00062013611.1701979120.226752549254N-acetylornithine0.829892374.192175.1070165Organic acids and derivativesForward0.0562798930.0470083061.8305870280.03537067211.1972329430.2597038822591-(Hydroxymethyl)-5,5-dimethyl-2,4-imidazolidinedione0.818992407.131159.0757679Organoheterocyclic compoundsForward0.1820869830.155835561.9456649270.01444207511.1684559280.224603319291Biochanin A0.76976639.59165285.07362Phenylpropanoids and polyketidesforward0.0317217030.0417325311.9202547120.01825747810.760119332−0.395702167295Heptadecanoyl carnitine0.756939194.733414.3571643Lipids and lipid-like moleculesForward0.0174031640.0221186512.1376965730.0007222710.786809469−0.3459137753153-[(3-Methylbutyl)nitrosoamino]-2-butanone0.71523136.73805187.1434871Organooxygen compoundsForward0.3399653070.4571208562.0613000250.00897733110.743709903−0.4271881123202-Acetylpyrazine0.710279330.908123.0549956Organooxygen compoundsForward0.0131233240.0087841521.9704703890.0202078611.4939773690.579158294328Sedoheptulose 1-phosphate0.70085440.022291.0477486Organic oxygen compoundsForward0.0510976910.0693458062.0465084060.00706959110.736853377−0.440550522360Glechomafuran0.646208224.3045231.1407859Lipids and lipid-like moleculesForward0.0100339710.0147501962.0959910590.00284462710.680260196−0.55584142384LysoPC(17 : 0)0.611195214.164510.3549507Lipids and lipid-like moleculesForward0.7720316391.0468688231.8899345340.04551365810.73746741−0.4393488410Acetyl-N-formyl-5-methoxykynurenamine0.57791624.7427265.1210007Organic oxygen compoundsForward0.0104525780.0064759942.189496149.71037E−0511.6140499980.690685269430Cysteinyl-serine0.56069125.0757209.0581161Organic acids and derivativesForward0.5428060050.2856816991.9417437440.04101055411.9000377260.926028064435L-octanoylcarnitine0.556023224.21288.2159451Lipids and lipid-like moleculesForward0.0644109740.0896772522.1069708410.00228456510.718253205−0.47743557442S-methylmethionine0.553207336.697164.0731609Forward0.0325733180.0448692741.8469457230.03290781810.725960445−0.4620371524714-Aminohippuric acid0.488799315.061184.0895643BenzenoidsForward0.0093285130.0230728761.9116708970.01025103610.404306452−1.306478867477PE(16 : 0/18 : 3(9Z,12Z,15Z))0.471196164.6285714.5059277Lipids and lipid-like moleculesForward0.0431752760.027338731.8381578390.03266264211.5792714560.659259173IDThe unique data number of the substance in this qualitative analysisMS2 nameThe substance name obtained by qualitative matching analysis of secondary mass spectrometryMS2 scoreThe score of the second-level match, with the value [0, 1]rtChromatographic retention time of the substancemzMaterial characteristics mass charge ratio of ionsSuperClassClassification information of the substance in the HMDB databaseTypeThe matching mode includes MS2 forward/reverse matching (MS2 forward/reverse), first-order average molecular weight/single isotope molecular weight matching and no matchingMEAN OOThe mean of the relative quantitative value of the substance in one of the experimental groups within the group comparisonMEAN XXThe mean of the relative quantitative value of the substance in the other experimental group within the group comparisonVIPThe substance in the group was compared to the OPLS-DA model obtained by the variable projection significanceP-valueThep-value obtained by the t-test of this substance in the group comparison, p-value = probabilityof hypothesis being correct but rejected = number of negative results/total number of results, is a test probability against the sample dataQ-valueThe hypothesis test statistic (p-value) is the result after the correction of multiple hypothesis test. Q value = the probability of being rejected but correct = the number of false-positive results/thenumber of presumed positive results. It is a test probability of the inference obtained from statistical test and the re-statistics of p-valueFOLD CHANGEThe substance in this group was compared with the multiple relationship between the two groups of experimentsLOG_FOLDCHANGEFOLD CHANGE takes the logarithm base 2Table 2
Differential metabolites detected by mass spectrometry combined with gas phase.
IDPeak2SimilarityrtCountMassMEAN TMEAN MVIPP-valueQ-valueFOLDCHANGELOG_FOLDCHANGE692-Hydroxybutanoic acid9235.90393,091311.2725150661.8291571.2998433990.0308247660.0547177460.695683796−0.523496376873-Hydroxybutyric acid9176.15207,0914725.4876259738.15091.4357728810.0069065930.0310583520.668074026−0.581920125228L-malic acid9108.38126,097315.0209830424.188151.2068502040.0458445990.0647071690.621005853−0.68732123407Palmitoleic acid90311.4327,09554.4277154536.2580291.2366459830.0496532090.0670783250.707525543−0.499145861410Palmitic acid87811.5306,0911720.9099871628.737531.3098507060.0250560750.050260230.727619379−0.45874412768Glycine 18765.88319,091023.9205308895.9694691.4371491150.0036231460.0310583520.656763806−0.606553473144glycerol8556.94956,0920514.7360911322.119351.3165077760.0303746470.0544071640.66620819−0.585955005234Threitol8528.47978,092170.3205982890.5348071.4397500270.0032849070.0310583520.599465913−0.738250374273Alpha-ketoglutaric acid8268.93667,09734.3927607567.0408611.190463660.049580740.0670351060.623895417−0.680623882172Uracil8227.43667,092410.3821254020.6614691.3530534570.0220607250.0474554370.577691894−0.791627843184Pelargonic acid7947.60111,092150.0463908640.075661.232558620.0438261990.0633623510.613151594−0.705684289222Aminomalonic acid7918.28837,092180.4134769310.5848891.2444331470.0313880310.0550986310.706932665−0.500355292062-Deoxytetronic acid7808.01104,09734.2877756646.9928051.3806595290.0077585490.0310583520.613169609−0.705641901853-Hydroxypyridine7786.0817,091520.2330751930.3495791.4734374490.0041902230.0310583520.666731544−0.5848221111274-Hydroxybutyrate7696.69919,092330.0183865990.0323411.340645620.0251107420.0503079060.568527482−0.814698006427heptadecanoic acid74511.985,091170.1105081210.1685021.3367032070.0320822580.0555565870.655828223−0.608610108197Thymine6777.85356,092700.0437783810.0661041.3716052110.0129065660.0378012160.662263926−0.59452182110Malonic acid 16746.46348,091410.1201531860.2125151.4809746110.0015763690.0310583520.56538798−0.82268688399Methyl phosphate6656.28585,092410.1156624740.2116871.4639365010.0027270240.0310583520.5463833−0.872014707207Beta-alanine 26438.03652,092480.0540565270.1013641.4210354910.0149506270.0396599960.533289189−0.9070100131622,3-Dihydroxypyridine6337.28778,092400.0864322910.129721.3933943510.0066638440.0310583520.666297891−0.585760767463N-acetyl-5-hydroxytryptamine 159013.2097,09290160.0062516231.71931.3030210850.0414787310.0617143610.690517501−0.5342501151133-Aminoisobutyric acid 25206.51726,09570.322001070.4964841.3389674690.0156377140.040208520.648562563−0.6246823453071,2-Cyclohexanedione 45179.492,082050.1017488130.1816781.3875714470.0266165590.0515765050.560051285−0.836369152216Erythrose 15148.18007,093500.6899316791.0920361.2724094680.0283014720.0529008680.631784782−0.662494907219Capric acid5088.24689,091170.1612120230.2745971.3585432350.0107270660.0353398220.587086555−0.7683548781462-Deoxyuridine5037.01356,03810.0002580020.6598531.5390405050.0161230390.0405760960.000390999−11.32054852377D-altrose 248710.8282,032570.0002580020.1383831.5418037310.0002791560.0310583520.0018644−9.067072758111Methylmalonic acid4816.46941,092260.2104692690.4239131.4962354510.0022031880.0310583520.496491996−1.010157634285Digitoxose 14529.08156,03730.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.589515221246trans-4-Hydroxy-L-proline 24498.64244,031860.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.58951522188Sulfuric acid4196.16896,092810.7595822161.1403431.3211894910.0300787260.0541998950.666099824−0.5861896952182,4-Diaminobutyric acid 34168.2337,091030.1773469780.2799411.3706080870.0091077190.0330745620.633516577−0.658545725425d-Glucoheptose 141211.9031,073190.0122581460.0210771.3154295160.0393163990.0603777040.581574962−0.781962934501Isomaltose 241015.118,07730.216801480.3308111.4627086030.0053479210.0310583520.655363457−0.609632863211N-acetyl-L-leucine 24068.12433,08860.0586787620.0871621.4475791250.0111256310.0358348260.673218373−0.570853545309L-homoserine 33969.52289,062900.0521601150.0758241.4817961590.002094980.0310583520.687909761−0.539708769772-Ketovaleric acid 23935.97089,09890.1480174450.2483581.447203690.0043150570.0310583520.595983832−0.7466549022384-Acetamidobutyric acid 23878.55919,091740.2626814940.4144911.3043952180.0239233440.0492452540.633744092−0.658027703343Biuret 238510.0571,061710.1311124380.111931.2984911140.0387837810.0600351661.1713794590.228208501149O-acetylserine 23847.08467,031160.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.589515221170Oxamide3827.37756,091160.6380630280.9637431.3750597840.0109468650.0356155660.66206751−0.594949762193Resorcinol3647.74837,092400.18937030.2705611.4221353430.0288400310.0533046360.699917794−0.514742608342-Ketobutyric acid 23595.37696,091743.8953987136.3109691.4527537710.0038196660.0310583520.617242587−0.69609049495Maltose34414.4673,032040.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.589515221506Ergosterol33917.6037,071290.0381424770.1700621.1520560640.0404762470.0611051750.224285234−2.156593449290D-erythronolactone 23389.18748,092440.2335205670.3354041.3387417370.0152381130.039893680.696237385−0.522348811417Sinapyl alcohol 133111.7691,032410.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.589515221416N-acetyl-D-galactosamine 132911.7478,032020.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.5895152211143-Methylamino-1,2-propanediol 13276.52733,091160.1459287130.2519511.4877100760.0005752240.0310583520.579194579−0.787879996924-Hydroxypyridine3266.20052,091410.4899277930.7959271.4513239710.0024659510.0310583520.615543839−0.700066486251Cytosine3228.68941,093142.5098405524.0820561.2831599780.0219633170.0473573920.614847194−0.701700187507Cholestane-3,5,6-triol, (3?5?6?-32118.7661,09570.8851466532.0021861.3875648920.0069055780.0310583520.442090187−1.1775873842952-Carboxybenzaldehyde3189.29889,034480.0002580020.0405661.541941440.0018885660.0310583520.006360115−7.296731326486Alizarin29613.9349,031920.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.589515221255Maleamate 22728.72638,072440.0179845420.0832381.1339189220.0230974270.0484710180.216061427−2.2104865641694-Acetylbutyric acid 22657.37059,09710.8804394141.4448541.4367706410.0187648950.0438594070.60936233−0.7146277771Analyte 104.85415,092200.1436275880.2069941.4125125280.0093754160.033479910.693874841−0.5272526372Analyte 204.86659,092210.9391797891.4825621.2231874220.0485398460.0664064660.633484237−0.6586193734Analyte 404.906,0912725.0483889140.416221.4272777130.0240226420.0493363510.619760853−0.6902164649Analyte 904.95133,09560.0760087240.1305491.3871058780.0064814080.0310583520.582225673−0.78034963913Analyte 1305.01281,092810.5191585430.881591.4159562770.004327660.0310583520.588888971−0.76393244114Analyte 1505.05415,09890.1266863660.1943851.3707323850.0113142690.0360614850.651727984−0.61765815315Analyte 1605.06096,09710.142475830.2545961.3824156910.0251283130.0503232040.559614273−0.83749533616Analyte 1705.10467,092480.1892362390.2774121.1858621230.0463494550.0650336210.682149873−0.55183935117Analyte 1805.1217,09710.235375970.3690891.2852373470.0282230690.0528413250.63772183−0.64900082821Analyte 2205.17593,091600.1173364380.2273761.3967641480.0147131460.0394622340.516045471−0.95442990226Analyte 2705.21607,092071.5341230142.4423951.4067900330.0051169320.0310583520.628122392−0.67088239428Analyte 2905.28378,093180.4905830820.816121.4367950450.0033191470.0310583520.601116114−0.734284429Analyte 3005.28985,091410.4241924950.6995251.4238410190.0233623250.0487226040.606400455−0.7216572631Analyte 3205.306,09820.1138901230.1580431.3007670640.0410701670.0614682230.720626315−0.47267675935Analyte 3605.38238,072650.0966598410.1409971.3235885580.0263448320.0513537720.685547745−0.54467094942Analyte 4305.50956,063180.0531479620.1166361.3890437740.0049726260.0310583520.45567417−1.13392550243Analyte 4405.53119,09831.0926466831.8996171.3748646830.0144040960.0391982180.575193161−0.79788157245Analyte 4605.57133,091730.0406811950.0619561.2805314430.0325395280.0558515340.656616292−0.60687754753Analyte 5405.69844,03720.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.58951522160Analyte 6105.77652,091870.4117390970.6407321.3818764880.0342612640.0569169130.642607665−0.63798990661Analyte 6205.78067,09890.1962076650.2807791.2497140740.0324071580.0557666890.698796716−0.51705526765Analyte 6605.85193,092581.1027398691.8985711.459403240.0028411140.0310583520.58082638−0.78382111666Analyte 6705.86748,092450.751411871.0420031.3408555380.0147209080.0394687670.721122764−0.47168320970Analyte 7105.91167,08830.1246001320.210661.4601880.0059234320.0310583520.5914747−0.75761163671Analyte 7205.917,082060.1098841710.152681.265734030.0412871140.0615992840.719702949−0.47452652679Analyte 8005.97948,091300.065456180.1038261.4575718180.0026013190.0310583520.630443919−0.66556005283Analyte 8406.06822,09890.6298836150.8571531.3489649080.021462270.0468456660.734855621−0.44446726686Analyte 8706.1057,093550.0131006010.0233431.4648594920.0029202620.0310583520.561219926−0.83336186394Analyte 9506.23356,093220.0910504220.1412991.3359358170.0130503720.0379445490.644383212−0.63400918896Analyte 9706.25607,092210.2733344860.5051441.3827182810.0184611580.0434964260.541101922−0.8860277397Analyte 9806.26304,092670.0521838010.0813531.3390446480.0218878510.0472811140.641446738−0.64059861798Analyte 9906.28007,091521.2432812841.7312471.3624503450.0181108880.043070520.718142143−0.477658667100Analyte 10106.35356,092110.1499751120.2432341.2694344250.0344425560.0570251210.6165884−0.697620347101Analyte 10206.37207,092210.1260686150.2297191.3698120920.0149189150.0396338390.548793895−0.865663663112Analyte 11406.51,032070.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.589515221124Analyte 12606.667,041590.0059801180.0353981.2067438520.0075958190.0310583520.168941731−2.565402355132Analyte 13406.76096,091020.0871561260.131351.4244133830.0046944750.0310583520.663543315−0.591737451135Analyte 13706.83089,091080.1974753330.3222811.3940145940.0094837190.0336400960.612743258−0.70664539138Analyte 14006.86778,093191.8731576393.1141221.4183622990.0078473030.0310583520.601504164−0.73335337147Analyte 14907.02956,09299230.5117654328.73471.3685011320.0144506120.0392384470.70120909−0.512083397150Analyte 15207.08911,031080.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.589515221168Analyte 17007.36319,092210.0386756130.0643061.4252992040.0037797370.0310583520.601426746−0.733539068174Analyte 17607.44822,031740.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.589515221176Analyte 17807.4663,093000.140669290.254311.3977994840.0093902640.0335019990.553141309−0.854280007183Analyte 18507.57178,092410.0346598520.053531.2789094920.0226391210.0480282390.647485316−0.62708062188Analyte 19007.65457,071520.0071840550.0273391.1175067830.0480282230.0660919740.262776377−1.92809251210Analyte 21208.07785,093270.0580094660.1015891.3813210630.0087969010.0325863550.571020535−0.808385465213Analyte 21508.15637,093020.0642571010.0987221.2462777130.0280986540.0527464330.650889175−0.619516174215Analyte 21708.16556,092810.0393451220.0804911.3749052610.0036862170.0310583520.48881664−1.032634697227Analyte 22908.36156,093410.0524487590.0869461.3252188160.0170928860.0417861740.603232561−0.72921379229Analyte 23108.40367,081000.2031800310.329391.3419799580.0136931480.0385605360.616837794−0.697036932237Analyte 24008.54081,093370.250365220.4556551.3667772460.0368323950.0587320650.549461995−0.863908397253Analyte 25608.70644,092920.3575289290.5159311.219381210.0484530830.0663533910.692978537−0.529117425256Analyte 25908.74333,082450.0488457080.0908291.3266294340.0097239220.0339878290.537777275−0.894919303259Analyte 26208.78319,092630.130882510.2310691.3448605660.0444344420.0637744450.566422421−0.820049721267Analyte 27008.88467,09710.112272970.2041381.4330632660.006086670.0310583520.549984911−0.862536055345Analyte 350010.0673,052920.0002580020.0637221.5396282120.0049526840.0310583520.004048846−7.948273624364Analyte 369010.4758,03550.0002580020.0635521.5422249230.0162541120.0406727060.004059669−7.944422022393Analyte 400011.1802,062170.0617136490.1615871.4056551090.0135228450.0384011380.38192255−1.38864799435Analyte 442012.219,04670.0056926750.0304991.1844972070.0394247710.0604467360.186653558−2.421565088455Analyte 463012.8955,092323.4423798154.6092311.3595474290.0253858680.0505460710.746844736−0.421119747487Analyte 495013.9389,062590.0136908520.0270191.4015705030.0231381990.0485099450.506703381−0.98078664503Analyte 511015.8464,093570.0805725970.1451651.2708592360.0356178610.0578806230.555041297−0.849332977IDThe unique data number of the substance in this qualitative analysisPeakThe name of the substance obtained by qualitative analysisSimilarityThe matching degree between the substance and the substance in the standard library in qualitative analysis, and the value is [0, 1000]rtChromatographic retention time of the substanceCountThe number of times the substance was detected in all experimental groupsMassMaterial characteristics Mass charge ratio of ionsMEAN OOThe mean of the relative quantitative value of the substance in one of the experimental groups within the group comparisonMEAN XXThe mean of the relative quantitative value of the substance in the other experimental group within the group comparisonVIPThe substance in the group was compared to the OPLS-DAmodel obtained by the variable projection significanceP-valueThep-value obtained by the t-test of this substance in the group comparison, p-value = probability of hypothesis being correct but rejected = number of negative results/total number of results, is a test probability against the sample dataQ-valueThe hypothesis test statistic (p-value) is the result after the correction of multiple hypothesis test. Q-value = the probability of being rejected but correct = the number of false-positive results/the number of presumed positive results. It is a test probability of the inference obtained from statistical test and the re-statistics of p-valueFOLD CHANGEThe substance in this group was compared with the multiple relationship between the two groups of experimentsLOG_FOLDCHANGEFOLD CHANGE takes the logarithm base 2Figure 2
OPLS-DA model score scatter plot and differential metabolite analysis. (a) Scatter plot of OPLS-DA model score and volcano plot of differential metabolites for group M versus group T detected by mass spectrometry coupled with the high-performance liquid phase. (b) Scatter plot of OPLS-DA model score and volcano plot of differential metabolites for group M versus group T detected by mass spectrometry coupled with gas phase. The size of scattering in the volcano plot represents the VIP value of OPLS-DA model, the larger the scatter the larger the VIP value. Significantly upregulated metabolites are shown in red, significantly down-regulated metabolites are shown in blue, and nonsignificantly different metabolites are shown in gray. (c) Scatter plot of OPLS-DA model scores for group M versus group T detected by NMR. (d) OPLS-DA loadings plot for group M versus group (T). The horizontal coordinates in the plot indicate the magnitude of chemical shifts, from right to left, with increasing values of chemical shifts; the vertical coordinates indicate the magnitude of loadings after back conversion, where the shades of color indicate the magnitude of correlation coefficients, red indicates a positive correlation, and blue indicates negative correlation. The marked substances in the figure are the differential metabolites that passed the critical value test of the correlation coefficient.
(a)(b)(c)(d)


### 3.3. Enrichment Analysis of Metabolic Pathways of Differential Metabolites

We further analyzed the effect of differential metabolites on their pathways. The analysis based on the mass spectrometry combined with high-performance liquid phase detection results showed that the differential metabolites in the M and T groups were enriched to 11 pathways (Table3), among which the pathways with higher enrichment were histidine metabolism, amino acid tRNA biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, tryptophan metabolism, phenylalanine metabolism, cysteine, and methionine metabolism. The analysis based on the results of mass spectrometry combined with gas-phase detection showed that the differential metabolites of the M and T groups were enriched to 28 pathways (Table 4), among which the pathways with higher enrichment were pyrimidine metabolism, biosynthesis of pantothenic acid and CoA, metabolism of β-alanine, sulfur metabolism, glycerolipid metabolism, and metabolism of glycine, serine, and threonine. Subsequently, we took the intersection of the pathways enriched by the differential metabolites in the M and T groups for both high-performance liquid- and gas-phase methods coupled to mass spectrometry and obtained a total of five common pathways (biosynthesis of amino acid tRNA, metabolism of D-glutamine and D-glutamate, metabolism of tryptophan, metabolism of nitrogen, and metabolism of arginine and proline). See Figure 3Table 3
Enrichment analysis results based on mass spectrometry combined with HPLC.
PathwayTotalHitsRawp−ln (p)Holm adjustFDRImpactHits cpdTotal cpdHistidinemetabolism1520.0115264.46320.950.945120.24194L-Histidine cpd:C00135; carnosine cpd: C00386L-glutamic acid cpd: C00025; 4-imidazolone-5-propionic acid cpd: C03680; urocanic acid cpd: C00785; L-histidine cpd: C00135; imidazole-4-acetaldehyde cpd: C05130; 1-methylhistamine cpd: C05127; methylimidazole acetaldehyde cpd: C05827; histamine cpd: C00388; carnosine cpd: C00386; N-formyl-L-aspartate cpd: C01044; formiminoglutamic acid cpd: C00439; imidazoleacetic acid cpd: C02835; methylimidazoleacetic acid cpd: C05828; L-aspartic acid cpd: C00049; 1-methylhistidine cpd: C01152Aminoacyl-tRNA biosynthesis6930.0392323.2383110L-histidine cpd: C00135; L-phenylalanine cpd: C00079; L-methionine cpd: C00073L-asparagine cpd: C00152; tRNA(Asn) cpd: C01637; L-histidine cpd: C00135; tRNA(His) cpd: C01643; L-phenylalanine cpd: C00079; tRNA(Phe) cpd: C01648; L-arginine cpd: C00062; tRNA(Arg) cpd: C01636; L-glutamine cpd: C00064; tRNA(Gln) cpd: C01640; L-cysteine cpd: C00097; tRNA(Cys) cpd: C01639; glycine cpd: C00037; tRNA(Gly) cpd: C01642; tRNA(Asp) cpd: C01638; L-aspartic acid cpd: C00049; L-serine cpd: C00065; tRNA(Ser) cpd: C01650; L-methionine cpd: C00073; tRNA(Met) cpd: C01647; L-valine cpd: C00183; tRNA(Val) cpd: C01653; L-alanine cpd: C00041; tRNA(Ala) cpd: C01635; L-lysine cpd: C00047; tRNA(Lys) cpd: C01646; L-isoleucine cpd: C00407; tRNA(Ile) cpd: C01644; tRNA(Leu) cpd: C01645; L-leucine cpd: C00123; L-threonine cpd: C00188; tRNA(Thr) cpd: C01651; tRNA(Trp) cpd: C01652; L-tryptophan cpd: C00078; N10-formyl-thf cpd: C00234; L-methionyl-tRNA cpd: C02430; L-tyrosine cpd: C00082; tRNA(Tyr) cpd: C00787; L-proline cpd: C00148; tRNA(Pro) cpd: C01649; L-glutamic acid cpd: C00025; tRNA(Glu) cpd: C01641; glutaminyl-tRNA cpd: C02282; L-asparaginyl-tRNA(Asn) cpd: C03402; tRNA(Sec) cpd: C16636; L-seryl-tRNA(Sec) cpd: C06481; O-phosphoseryl-tRNA(Sec) cpd: C16638; L-histidyl-tRNA(His) cpd: C02988; L-phenylalanyl-tRNA(Phe) cpd: C03511; L-arginyl-tRNA(Arg) cpd: C02163; L-cysteinyl-tRNA(Cys) cpd: C03125; glycyl-tRNA(Gly) cpd: C02412; L-aspartyl-tRNA(Asp) cpd: C02984; L-seryl-tRNA(Ser) cpd: C02553; L-valyl-tRNA(Val) cpd: C02554; L-alanyl-tRNA cpd: C00886; L-lysyl-tRNA cpd: C01931; L-isoleucyl-tRNA(Ile) cpd: C03127; L-leucyl-tRNA cpd: C02047; L-threonyl-tRNA(Thr) cpd: C02992; L-tryptophanyl-tRNA(Trp) cpd: C03512; tetrahydrofolic acid cpd: C00101; N-formylmethionyl-tRNA cpd: C03294; L-tyrosyl-tRNA(Tyr) cpd: C02839; L-prolyl-tRNA(Pro) cpd: C02702; L-glutamyl-tRNA(Glu) cpd: C02987; L-glutamyl-tRNA(Gln) cpd: C06112; L-aspartyl-tRNA(Asn) cpd: C06113; L-selenocysteinyl-tRNA(Sec) cpd: C06482Phenylalanine, tyrosine, and tryptophan biosynthesis410.0444533.1133110.5L-phenylalanine cpd: C00079Phenylpyruvic acid cpd: C00166; L-phenylalanine cpd: C00079; L-tyrosine cpd: C00082; 4-hydroxyphenylpyruvic acid cpd: C01179D-glutamine and D-glutamate metabolism510.0552732.8955110D-glutamine cpd: C00819L-glutamic acid cpd: C00025; D-glutamine cpd: C00819; L-glutamine cpd: C00064; oxoglutaric acid cpd: C00026; D-glutamic acid cpd: C00217Tryptophan metabolism4020.0726662.6219110.10987L-kynurenine cpd: C00328; acetyl-N-formyl-5-methoxykynurenamine cpd: C05642L-tryptophan cpd: C00078; melatonin cpd: C01598; serotonin cpd: C00780; 5-hydroxykynurenamine cpd: C05638; 5-hydroxykynurenine cpd: C05651; 5-hydroxy-L-tryptophan cpd: C00643; L-formylkynurenine cpd: C02700; acetoacetyl-CoA cpd: C00332; (S)-3-hydroxybutanoyl-CoA cpd: C01144; crotonoyl-CoA cpd: C00877; glutaryl-CoA cpd: C00527; oxoadipic acid cpd: C00322; 2-amino-3-carboxymuconic acid semialdehyde cpd: C04409; 3-hydroxyanthranilic acid cpd: C00632; L-kynurenine cpd: C00328; formylanthranilic acid cpd: C05653; L-3-hydroxykynurenine cpd: C03227; 3-hydroxykynurenamine cpd: C05636; indoleacetaldehyde cpd: C00637; 5-hydroxy-N-formylkynurenine cpd: C05648; 5-hydroxyindoleacetaldehyde cpd: C05634; tryptamine cpd: C00398; indoleacrylic acid cpd: C00331; acetyl-N-formyl-5-methoxykynurenamine cpd: C05642; 6-hydroxymelatonin cpd: C05643; N-acetylserotonin cpd: C00978; formyl-5-hydroxykynurenamine cpd: C05647; 4,6-dihydroxyquinoline cpd: C05639; acetyl-CoA cpd: C00024; 2-aminomuconic acid semialdehyde cpd: C03824; 2-aminobenzoic acid cpd: C00108; L-tryptophanyl-tRNA(Trp) cpd: C03512; cinnavalininate cpd: C05640; 4-(2-amino-3-hydroxyphenyl)-2,4-dioxobutanoic acid cpd: C05645; 4-(2-aminophenyl)-2,4-dioxobutanoic acid cpd: C01252; 4,8-dihydroxyquinoline cpd: C05637; indoleacetic acid cpd: C00954; 5-hydroxyindoleacetic acid cpd: C05635; N-methylserotonin cpd: C06212; N-methyltryptamine cpd: C06213Nitrogen metabolism910.0974152.3288110L-histidine cpd: C00135Ammonia cpd: C00014; carbon dioxide cpd: C00011; L-glutamic acid cpd: C00025; L-Glutamine cpd: C00064; L-cystathionine cpd: C02291; L-Histidine cpd: C00135; carbamoylphosphate cpd: C00169; carbonic acid cpd: C01353; glycine cpd: C00037Phenylalanine metabolism1110.117822.1386110.40741L-phenylalanine cpd: C00079Phenylacetaldehyde cpd: C00601; phenylacetyl-CoA cpd: C00582; L-phenylalanine cpd: C00079; phenylethylamine cpd: C05332; phenylpyruvic acid cpd: C00166; phenylacetic acid cpd: C07086; phenylacetylglycine cpd: C05598; ortho-hydroxyphenylacetic acid cpd: C05852; enol-phenylpyruvate cpd: C02763; 2-phenylacetamide cpd: C02505; L-tyrosine cpd: C00082Sphingolipid metabolism2110.213511.5441110Phytosphingosine cpd: C12144Sphinganine cpd: C00836; ceramide 1-phosphate cpd: C02960; sphingosine 1-phosphate cpd: C06124; sphinganine 1-phosphate cpd: C01120; dihydroceramide cpd: C12126; phytoceramide cpd: C12145; SM cpd: C00550; ceramide cpd: C00195; Palmityl-CoA cpd: C00154; L-Serine cpd: C00065; 3-dehydrosphinganine cpd: C02934; galabiosylceramide cpd: C06126; galactosylceramide cpd: C02686; GM4 cpd: C06128; lactosylceramide cpd: C01290; glucosylceramide cpd: C01190; sphingosine cpd: C00319; 3-O-Sulfogalactosylceramide (d18 :  : 1/24 :  : 0) cpd: C06125; phytosphingosine cpd: C12144; digalactosylceramidesulfate cpd: C06127; O-phosphoethanolamine cpd: C00346Cysteine and methionine metabolism2710.266161.3236110.08685L-methionine cpd: C000732-Oxo-4-methylthiobutanoic acid cpd: C01180; 1,2-dihydroxy-3-keto-5-methylthiopentene cpd: C15606; 5′-methylthioadenosine cpd: C00170; S-adenosylmethioninamine cpd: C01137; S-adenosylmethionine cpd: C00019; L-cystathionine cpd: C02291; L-homocysteine cpd: C00155; L-serine cpd: C00065; L-methionine cpd: C00073; S-adenosylhomocysteine cpd: C00021; 2,3-diketo-5-methylthiopentyl-1-phosphate cpd: C15650; cysteic acid cpd: C00506; L-cystine cpd: C00491; L-cysteine cpd: C00097; 3-sulfinoalanine cpd: C00606; sulfite cpd: C00094; 3-mercaptopyruvic acid cpd: C00957; 3-methylthiopropionic acid cpd: C08276; 5-methylthioribose 1-phosphate cpd: C04188; 2-ketobutyric acid cpd: C00109; 2-aminoacrylic acid cpd: C02218; 3-sulfopyruvic acid cpd: C05528; thiocysteine cpd: C01962; 3-sulfinylpyruvic acid cpd: C05527; pyruvic acid cpd: C00022; thiosulfate cpd: C00320; 3-mercaptolactic acid cpd: C05823Glycerophospholipid metabolism3010.291231.2336110.02315Glycerophosphocholine cpd: C00670Phosphatidylethanolamine cpd: C00350; phosphatidylcholine cpd: C00157; dihydroxyacetone phosphate cpd: C00111; LysoPC (18 : 1(9Z)) cpd: C04230; 1,2-diacyl-sn-glycerol cpd: C00641; citicoline cpd: C00307; phosphorylcholine cpd: C00588; choline cpd: C00114; acetylcholine cpd: C01996; O-phosphoethanolamine cpd: C00346; ethanolamine cpd: C00189; PA (16 :  : 0/16 :  : 0) cpd: C00416; acyl-CoA cpd: C00040; 1-acyl-sn-glycerol 3-phosphate cpd: C00681; CDP-diacylglycerol cpd: C00269; glycerol 3-phosphate cpd: C00093; 1-acyl-sn-glycero-3-phosphoethanolamine cpd: C04438; 2-acyl-sn-glycero-3-phosphoethanolamine cpd: C05973; 2-acyl-sn-glycero-3-phosphocholine cpd: C04233; CDP-glycerol cpd: C00513; PS (16 : 0/16 : 0) cpd: C02737; phosphatidylglycerol cpd: C00344; dihydroxyacetone phosphate acyl ester cpd: C03372; CDP-ethanolamine cpd: C00570; 1-phosphatidyl-D-myo-inositol cpd: C01194; glycerylphosphorylethanolamine cpd: C01233; glycerophosphocholine cpd: C00670; phosphatidyl-N-methylethanolamine cpd: C01241; phosphatidylglycerophosphate cpd: C03892; cardiolipin cpd: C05980Arginine and proline metabolism4410.397960.9214110N-acetylornithine cpd: C00437L-glutamine cpd: C00064; ammonia cpd: C00014; carbamoylphosphate cpd: C00169; ornithine cpd: C00077; citrulline cpd: C00327; L-aspartic acid cpd: C00049; argininosuccinic acid cpd: C03406; L-arginine cpd: C00062; L-glutamic acid cpd: C00025; N-acetylornithine cpd: C00437; L-proline cpd: C00148; peptide cpd: C00012; D-proline cpd: C00763; hydroxyproline cpd: C01157; pyrroline hydroxycarboxylic acid cpd: C04281; L-4-hydroxyglutamate semialdehyde cpd: C05938; L-erythro-4-hydroxyglutamate cpd: C05947; N-(o)-hydroxyarginine cpd: C05933; guanidinoacetic acid cpd: C00581; creatine cpd: C00300; gamma-aminobutyric acid cpd: C00334; agmatine cpd: C00179; L-glutamic-gamma-semialdehyde cpd: C01165; L-glutamic acid 5-phosphate cpd: C03287; (S)-1-pyrroline-5-carboxylate cpd: C03912; putrescine cpd: C00134; 4-aminobutyraldehyde cpd: C00555; S-adenosylmethioninamine cpd: C01137; S-adenosylmethionine cpd: C00019; spermidine cpd: C00315; N-acetylputrescine cpd: C02714; N4-acetylaminobutanal cpd: C05936; cis-4-hydroxy-D-proline cpd: C03440; fumaric acid cpd: C00122; urea cpd: C00086; N-acetyl-L-alanine cpd: C00624; 1-pyrroline-2-carboxylic acid cpd: C03564; D-4-hydroxy-2-oxoglutarate cpd: C05946; nitric oxide cpd: C00533; phosphocreatine cpd: C02305; 4-guanidinobutanoic acid cpd: C01035; spermine cpd: C00750; 4-acetamidobutanoic acid cpd: C02946; 1-pyrroline-4-hydroxy-2-carboxylate cpd: C04282PathwayMetabolic pathway nameTotalThe number of metabolites in this pathwayHitsThe number of differential metabolites hit this pathwayRawpP value of metabolic pathway enrichment analysis−ln(p)Minus log base E of PHolm adjustP values corrected by Holm–Bonferroni method for multiple hypothesis testingFDRP value corrected by false discovery rate (FDR) method for multiple hypothesis testingImpactImpact value of metabolic pathway topology analysisHits cpdThe names and KEGG IDs of differential metabolites hit the pathwayTotal cpdAll metabolite names and KEGG IDs contained in this pathwayTable 4
Enrichment analysis results based on mass spectrometry combined with gas phase.
PathwayTotalHitsRawp−ln(p)Holm adjustFDRImpactHits cpdTotal cpdPyrimidine metabolism4150.00136.62930.110.1080.09731Deoxyuridine cpd: C00526; uracil cpd: C00106; beta-alanine cpd: C00099; thymine cpd: C00178; 3-aminoisobutanoic acid cpd: C05145Thioredoxin cpd: C00342; uridine 5′-diphosphate cpd: C00015; L-Glutamine cpd: C00064; carbamoylphosphate cpd: C00169; 4,5-dihydroorotic acid cpd: C00337; orotidylic acid cpd: C01103; RNA cpd: C00046; uridine triphosphate cpd: C00075; uridine 5′-monophosphate cpd: C00105; uridine cpd: C00299; dihydrouracil cpd: C00429; ureidopropionic acid cpd: C02642; cytidine triphosphate cpd: C00063; CDP cpd: C00112; cytidine monophosphate cpd: C00055; cytidine cpd: C00475; thioredoxin disulfide cpd: C00343; dCDP cpd: C00705; dCTP cpd: C00458; dCMP cpd: C00239; deoxycytidine cpd: C00881; deoxyuridine triphosphate cpd: C00460; dUDP cpd: C01346; dUMP cpd: C00365; deoxyuridine cpd: C00526; thymidine 5′-triphosphate cpd: C00459; dTDP cpd: C00363; 5-thymidylic acid cpd: C00364; thymidine cpd: C00214; dihydrothymine cpd: C00906; ureidoisobutyric acid cpd: C05100; P1,P4-bis(5′-uridyl) tetraphosphate cpd: C06198; ureidosuccinic acid cpd: C00438; phosphoribosyl pyrophosphate cpd: C00119; orotic acid cpd: C00295; uracil cpd: C00106; beta-alanine cpd: C00099; DNA cpd: C00039; deoxyribose 1-phosphate cpd: C00672; thymine cpd: C00178; 3-aminoisobutanoic acid cpd: C05145Pantothenate and CoA biosynthesis1520.03843.2608110Beta-alanine cpd: C00099; uracil cpd: C00106Dephospho-CoA cpd: C00882; pantetheine 4′-phosphate cpd: C01134; pantetheine cpd: C00831; 4-phosphopantothenoylcysteine cpd: C04352; D-pantothenoyl-L-cysteine cpd: C04079; d-4′-phosphopantothenate cpd: C03492; L-cysteine cpd: C00097; pantothenic acid cpd: C00864; ureidopropionic acid cpd: C02642; dihydrouracil cpd: C00429; L-valine cpd: C00183; coenzyme a cpd: C00010; beta-alanine cpd: C00099; uracil cpd: C00106; alpha-ketoisovaleric acid cpd: C00141Beta-alanine metabolism1720.04843.0283110.44444Beta-alanine cpd: C00099; uracil cpd: C00106Acrylyl-CoA cpd: C00894; 3-hydroxypropionyl-CoA cpd: C05668; hydroxypropionic acid cpd: C01013; malonyl-CoA cpd: C00083; beta-alanine cpd: C00099; L-aspartic acid cpd: C00049; spermine cpd: C00750; 1,3-diaminopropane cpd: C00986; 3-aminopropionaldehyde cpd: C05665; ureidopropionic acid cpd: C02642; dihydrouracil cpd: C00429; anserine cpd: C01262; propionyl-CoA cpd: C00100; Acetyl-CoA cpd: C00024; malonic semialdehyde cpd: C00222; spermidine cpd: C00315; uracil cpd: C00106Starch and sucrose metabolism1920.05932.8253110.07451D-maltose cpd: C00208; isomaltose cpd: C00252beta-D-fructose cpd: C02336; sucrose cpd: C00089; uridine diphosphate glucuronic acid cpd: C00167; uridine diphosphate glucose cpd: C00029; glucose 1-phosphate cpd: C00103; 3-methoxy-4-hydroxyphenylglycol glucuronide cpd: C03033; glucose 6-phosphate cpd: C00668; alpha-D-glucose cpd: C00267; starch cpd: C00369; amylose cpd: C00718; trehalose cpd: C01083; D-maltose cpd: C00208; D-glucose cpd: C00031; dextrin cpd: C00721; isomaltose cpd: C00252; beta-D-fructose 6-phosphate cpd: C05345; UDP-D-xylose cpd: C00190; alpha-D-glucose 1,6-bisphosphate cpd: C01231; D-glucuronic acid cpd: C00191Propanoate metabolism2020.0652.733110Beta-alanine cpd: C00099; 2-hydroxybutyric acid cpd: C05984(S)-methylmalonic acid semialdehyde cpd: C06002; methylmalonyl-CoA cpd: C00683; propionyl-CoA cpd: C00100; propinol adenylate cpd: C05983; R-methylmalonyl-CoA cpd: C01213; succinic acid cpd: C00042; hydroxypropionic acid cpd: C01013; beta-alanine cpd: C00099; 2-propyn-1-al cpd: C05985; acetyl-CoA cpd: C00024; malonyl-CoA cpd: C00083; 2-hydroxybutyric acid cpd: C05984; acrylyl-CoA cpd: C00894; propionic acid cpd: C00163; succinyl-CoA cpd: C00091; 3-hydroxypropionyl-CoA cpd: C05668; malonic semialdehyde cpd: C00222; propynoic acid cpd: C00804; acetoacetyl-CoA cpd: C00332; 2-ketobutyric acid cpd: C00109D-glutamine and D-glutamate metabolism510.10162.2866110Oxoglutaric acid cpd: C00026L-glutamic acid cpd: C00025; D-glutamine cpd: C00819; L-glutamine cpd: C00064; oxoglutaric acid cpd: C00026; D-glutamic acid cpd: C00217Sulfur metabolism510.10162.2866110.3Sulfate cpd: C00059Phosphoadenosine phosphosulfate cpd: C00053; adenosine phosphosulfate cpd: C00224; sulfite cpd: C00094; sulfate cpd: C00059; adenosine 3′,5′-diphosphate cpd: C00054Cyanoamino acid metabolism610.12072.1145110Glycine cpd: C000373-Cyano-L-alanine cpd: C02512; beta-aminopropionitrile cpd: C05670; glycine cpd: C00037; gamma-glutamyl-beta-cyanoalanine cpd: C05711; gamma-glutamyl-beta-aminopropiononitrile cpd: C06114; L-serine cpd: C00065Methane metabolism910.17561.7394110Glycine cpd: C00037S-formylglutathione cpd: C01031; glycine cpd: C00037; 5,10-methylene-THF cpd: C00143; S-(Hydroxymethyl)glutathione cpd: C14180; methanol cpd: C00132; formic acid cpd: C00058; L-serine cpd: C00065; formaldehyde cpd: C00067; 5-methyltetrahydrofolic acid cpd: C00440Nitrogen metabolism910.17561.7394110Glycine cpd: C00037Ammonia cpd: C00014; carbon dioxide cpd: C00011; L-glutamic acid cpd: C00025; L-glutamine cpd: C00064; L-cystathionine cpd: C02291; L-histidine cpd: C00135; carbamoylphosphate cpd: C00169; carbonic acid cpd: C01353; glycine cpd: C00037Arginine and proline metabolism4420.23821.4345110.04414Hydroxyproline cpd: C01157; 4-acetamidobutanoic acid cpd: C02946L-glutamine cpd: C00064; ammonia cpd: C00014; carbamoylphosphate cpd: C00169; ornithine cpd: C00077; citrulline cpd: C00327; L-aspartic acid cpd: C00049; argininosuccinic acid cpd: C03406; L-arginine cpd: C00062; L-glutamic acid cpd: C00025; N-acetylornithine cpd: C00437; L-proline cpd: C00148; peptide cpd: C00012; D-proline cpd: C00763; hydroxyproline cpd: C01157; pyrroline hydroxycarboxylic acid cpd: C04281; L-4-hydroxyglutamate semialdehyde cpd: C05938; L-erythro-4-hydroxyglutamate cpd: C05947; N-(o)-hydroxyarginine cpd: C05933; guanidinoacetic acid cpd: C00581; creatine cpd: C00300; gamma-aminobutyric acid cpd: C00334; agmatine cpd: C00179; L-glutamic-gamma-semialdehyde cpd: C01165; L-glutamic acid 5-phosphate cpd: C03287; (S)-1-pyrroline-5-carboxylate cpd: C03912; putrescine cpd: C00134; 4-aminobutyraldehyde cpd: C00555; S-adenosylmethioninamine cpd: C01137; S-adenosylmethionine cpd: C00019; spermidine cpd: C00315; N-Acetylputrescine cpd: C02714; N4-Acetylaminobutanal cpd: C05936; cis-4-hydroxy-D-proline cpd: C03440; Fumaric acid cpd: C00122; urea cpd: C00086; N-Acetyl-L-alanine cpd: C00624; 1-pyrroline-2-carboxylic acid cpd: C03564; D-4-hydroxy-2-oxoglutarate cpd: C05946; nitric oxide cpd: C00533; phosphocreatine cpd: C02305; 4-guanidinobutanoic acid cpd: C01035; spermine cpd: C00750; 4-acetamidobutanoic acid cpd: C02946; 1-pyrroline-4-hydroxy-2-carboxylate cpd: C04282Glycerolipid metabolism1810.32131.1355110.28098Glycerol cpd: C00116Triacylglycerol cpd: C00422; acyl-CoA cpd: C00040; 1,2-diacyl-sn-glycerol cpd: C00641; PA(16 :  : 0/16 :  : 0) cpd: C00416; 1-acyl-sn-glycerol 3-phosphate cpd: C00681; 1-acylglycerol cpd: C01885; glycerol 3-phosphate cpd: C00093; dihydroxyacetone cpd: C00184; glycerol cpd: C00116; lactaldehyde cpd: C05999; D-glyceraldehyde cpd: C00577; glyceric acid cpd: C00258; digalactosyl-diacylglycerol cpd: C06037; fatty acid cpd: C00162; dihydroxyacetone phosphate cpd: C00111; propylene glycol cpd: C00583; 3-phospho-D-glycerate cpd: C00197; 1,2-diacyl-3-beta-D-galactosyl-sn-glycerol cpd: C03692Citrate cycle (TCA cycle)2010.35011.0496110.06799Oxoglutaric acid cpd: C00026Enzyme N6-(dihydrolipoyl)lysine cpd: C15973; oxoglutaric acid cpd: C00026; thiamine pyrophosphate cpd: C00068; Enzyme N6-(lipoyl)lysine cpd: C15972; 3-carboxy-1-hydroxypropylthiamine diphosphate cpd: C05381; Succinyl-CoA cpd: C00091; succinic acid cpd: C00042; oxalosuccinic acid cpd: C05379; isocitric acid cpd: C00311; oxalacetic acid cpd: C00036; Acetyl-CoA cpd: C00024; L-malic acid cpd: C00149; cis-aconitic acid cpd: C00417; citric acid cpd: C00158; pyruvic acid cpd: C00022; 2-(a-hydroxyethyl)thiamine diphosphate cpd: C05125; [dihydrolipoyllysine-residue succinyltransferase] S-succinyldihydrolipoyllysine cpd: C16254; Fumaric acid cpd: C00122; S-acetyldihydrolipoamide-E cpd: C16255; phosphoenolpyruvic acid cpd: C00074Butanoate metabolism2210.37770.97365110Oxoglutaric acid cpd: C000263-Butyn-1-al cpd: C06145; (R)-3-hydroxybutyric acid cpd: C01089; acetoacetic acid cpd: C00164; 3-hydroxy-3-methylglutaryl-CoA cpd: C00356; Acetyl-CoA cpd: C00024; Acetoacetyl-CoA cpd: C00332; (S)-3-hydroxybutanoyl-CoA cpd: C01144; Crotonoyl-CoA cpd: C00877; gamma-aminobutyric acid cpd: C00334; L-glutamic acid cpd: C00025; Butanoyl-CoA cpd: C00136; butanal cpd: C01412; succinic acid semialdehyde cpd: C00232; butyric acid cpd: C00246; oxoglutaric acid cpd: C00026; thiamine pyrophosphate cpd: C00068; pyruvic acid cpd: C00022; 3-butynoate cpd: C06144; 1-butanol cpd: C06142; succinic acid cpd: C00042; 2-hydroxyglutarate cpd: C02630; 2-(a-hydroxyethyl)thiamine diphosphate cpd: C05125Alanine, aspartate, and glutamate metabolism2410.40420.90587110.06329Oxoglutaric acid cpd: C00026N-Acetyl-L-aspartic acid cpd: C01042; 2-oxosuccinamate cpd: C02362; L-aspartic acid cpd: C00049; D-aspartic acid cpd: C00402; argininosuccinic acid cpd: C03406; adenylsuccinic acid cpd: C03794; L-Alanine cpd: C00041; succinic acid semialdehyde cpd: C00232; L-glutamic acid cpd: C00025; gamma-aminobutyric acid cpd: C00334; L-Glutamine cpd: C00064; ammonia cpd: C00014; 2-keto-glutaramic acid cpd: C00940; (S)-1-pyrroline-5-carboxylate cpd: C03912; oxalacetic acid cpd: C00036; L-Asparagine cpd: C00152; Fumaric acid cpd: C00122; pyruvic acid cpd: C00022; ureidosuccinic acid cpd: C00438; succinic acid cpd: C00042; oxoglutaric acid cpd: C00026; carbamoylphosphate cpd: C00169; glucosamine 6-phosphate cpd: C00352; 5-phosphoribosylamine cpd: C03090Galactose metabolism2610.42960.84494110Glycerol cpd: C00116Stachyose cpd: C01613; D-tagatose 6-phosphate cpd: C01097; D-gal alpha 1->6d-gal alpha 1->6d-glucose cpd: C05404; sucrose cpd: C00089; raffinose cpd: C00492; melibiose cpd: C05402; D-galactose cpd: C00124; galactosylglycerol cpd: C05401; epimelibiose cpd: C05400; melibiitol cpd: C05399; galactinol cpd: C01235; Alpha-D-Glucose cpd: C00267; alpha-lactose cpd: C00243; glucose 1-phosphate cpd: C00103; uridine diphosphategalactose cpd: C00052; uridine diphosphate glucose cpd: C00029; galactose 1-phosphate cpd: C00446; glucose 6-phosphate cpd: C00668; D-tagatose 1,6-bisphosphate cpd: C03785; D-Fructose cpd: C00095; D-glucose cpd: C00031; galactitol cpd: C01697; glycerol cpd: C00116; D-Mannose cpd: C00159; sorbitol cpd: C00794; myoinositol cpd: C00137Glutathione metabolism2610.42960.84494110.00573Glycine cpd: C00037R-S-cysteinylglycine cpd: C05729; R-S-glutathione cpd: C02320; glutathione cpd: C00051; NADP cpd: C00006; NADPH cpd: C00005; oxidized glutathione cpd: C00127; gamma-glutamylcysteine cpd: C00669; glycine cpd: C00037; L-cysteine cpd: C00097; L-glutamic acid cpd: C00025; cysteinylglycine cpd: C01419; pyroglutamic acid cpd: C01879; L-amino acid cpd: C00151; 5-L-glutamyl-L-alanine cpd: C03740; RX cpd: C01322; ornithine cpd: C00077; putrescine cpd: C00134; spermidine cpd: C00315; cadaverine cpd: C01672; tryparedoxin cpd: C16663; trypanothione cpd: C02090; S-substituted L-cysteine cpd: C05726; spermine cpd: C00750; aminopropylcadaverine cpd: C16565; tryparedoxin disulfide cpd: C16664; trypanothione disulfide cpd: C03170Fatty acid elongation in mitochondria2710.44190.8167110Palmitic acid cpd: C00249Palmityl-CoA cpd: C00154; (2e)-hexadecenoyl-CoA cpd: C05272; (S)-3-hydroxyhexadecanoyl-CoA cpd: C05258; 3-oxohexadecanoyl-CoA cpd: C05259; Acetyl-CoA cpd: C00024; Tetradecanoyl-CoA cpd: C02593; (2e)-tetradecenoyl-CoA cpd: C05273; (S)-3-hydroxytetradecanoyl-CoA cpd: C05260; 3-oxotetradecanoyl-CoA cpd: C05261; Lauroyl-CoA cpd: C01832; (2e)-dodecenoyl-CoA cpd: C03221; (S)-3-hydroxydodecanoyl-CoA cpd: C05262; 3-oxododecanoyl-CoA cpd: C05263; Decanoyl-CoA (n-C10: 0CoA) cpd: C05274; (2e)-decenoyl-CoA cpd: C05275; (S)-hydroxydecanoyl-CoA cpd: C05264; 3-oxodecanoyl-CoA cpd: C05265; Octanoyl-CoA cpd: C01944; (2e)-octenoyl-CoA cpd: C05276; (S)-hydroxyoctanoyl-CoA cpd: C05266; 3-oxooctanoyl-CoA cpd: C05267; Hexanoyl-CoA cpd: C05270; trans-2-hexenoyl-CoA cpd: C05271; (S)-hydroxyhexanoyl-CoA cpd: C05268; 3-oxohexanoyl-CoA cpd: C05269; Butanoyl-CoA cpd: C00136; palmitic acid cpd: C00249Porphyrin and chlorophyll metabolism2710.44190.8167110Glycine cpd: C00037Cobinamide cpd: C05774; heme O cpd: C15672; heme cpd: C00032; glycine cpd: C00037; bilirubin diglucuronide cpd: C05787; Cob(I)yrinate a,c diamide cpd: C06505; Fe2 + cpd: C14818; hemoglobin cpd: C01708; biliverdin cpd: C00500; protoporphyrinogen IX cpd: C01079; coproporphyrin III cpd: C03263; uroporphyrinogen III cpd: C01051; hydroxymethylbilane cpd: C01024; porphobilinogen cpd: C00931; 5-aminolevulinic acid cpd: C00430; uroporphyrinogen I cpd: C05766; L-Glutamic acid cpd: C00025; bilirubin cpd: C00486; protoporphyrin IX cpd: C02191; adenosyl cobinamide cpd: C06508; heme a cpd: C15670; D-Urobilinogen cpd: C05791; adenosyl cobyrinic acid a,c diamide cpd: C06506; Fe3+ cpd: C14819; coproporphyrinogen I cpd: C05768; l-glutamyl-tRNA(Glu) cpd: C02987; cytochrome c cpd: C00524Glycine, serine, and threonine metabolism3110.48860.71625110.26884Glycine cpd: C00037L-Serine cpd: C00065; choline cpd: C00114; glyceric acid cpd: C00258; betaine cpd: C00719; guanidinoacetic acid cpd: C00581; 3-phospho-D-glycerate cpd: C00197; dimethylglycine cpd: C01026; L-cystathionine cpd: C02291; glycine cpd: C00037; phosphoserine cpd: C01005; sarcosine cpd: C00213; 5,10-methylene-THF cpd: C00143; L-threonine cpd: C00188; lipoylprotein cpd: C02051; aminoacetone cpd: C01888; tetrahydrofolic acid cpd: C00101; S-aminomethyldihydrolipoylprotein cpd: C01242; dihydrolipoylprotein cpd: C02972; D-serine cpd: C00740; betaine aldehyde cpd: C00576; hydroxypyruvic acid cpd: C00168; creatine cpd: C00300; phosphohydroxypyruvic acid cpd: C03232; L-cysteine cpd: C00097; glyoxylic acid cpd: C00048; L-2-amino-3-oxobutanoic acid cpd: C03508; pyruvic acid cpd: C00022; carbon dioxide cpd: C00011; 5-aminolevulinic acid cpd: C00430; pyruvaldehyde cpd: C00546; ammonia cpd: C00014Valine, leucine, and isoleucine degradation3810.56140.57739110Methylmalonic acid cpd: C02170Enzyme N6-(lipoyl)lysine cpd: C15972; 2-methyl-1-hydroxybutyl-ThPP cpd: C15978; enzyme N6-(dihydrolipoyl)lysine cpd: C15973; 2-methyl-1-hydroxypropyl-ThPP cpd: C15976; 3-methyl-1-hydroxybutyl-ThPP cpd: C15974; acetyl-CoA cpd: C00024; acetoacetyl-CoA cpd: C00332; acetoacetic acid cpd: C00164; 3-hydroxy-3-methylglutaryl-CoA cpd: C00356; 3-methylcrotonyl-CoA cpd: C03069; 3-hydroxyisovaleryl-CoA cpd: C05998; isovaleryl-CoA cpd: C02939; 3-methyl-2-oxovaleric acid cpd: C00671; thiamine pyrophosphate cpd: C00068; L-valine cpd: C00183; 2-methylacetoacetyl-CoA cpd: C03344; (S)-3-hydroxyisobutyrate cpd: C06001; tiglyl-CoA cpd: C03345; butyryl-CoA cpd: C00630; S-(2-methylbutanoyl)-dihydrolipoamide cpd: C15979; alpha-ketoisovaleric acid cpd: C00141; L-isoleucine cpd: C00407; R-methylmalonyl-CoA cpd: C01213; methylmalonyl-CoA cpd: C00683; propionyl-CoA cpd: C00100; (S)-methylmalonic acid semialdehyde cpd: C06002; (S)-b-aminoisobutyric acid cpd: C03284; 2-methyl-3-hydroxybutyryl-CoA cpd: C04405; (S)-3-hydroxyisobutyryl-CoA cpd: C06000; methacrylyl-CoA cpd: C03460; (S)-2-methylbutanoyl-CoA cpd: C15980; S-(2-methylpropionyl)-dihydrolipoamide-E cpd: C15977; 4-methyl-2-oxopentanoate cpd: C00233; S-(3-methylbutanoyl)-dihydrolipoamide-E cpd: C15975; L-leucine cpd: C00123; 3-methylglutaconyl-CoA cpd: C03231; succinyl-CoA cpd: C00091; methylmalonic acid cpd: C02170Fatty acid metabolism3910.57090.56053110Palmitic acid cpd: C00249Palmityl-CoA cpd: C00154; hexanoyl-CoA cpd: C05270; butanoyl-CoA cpd: C00136; acetyl-CoA cpd: C00024; (S)-3-hydroxybutanoyl-CoA cpd: C01144; cis,cis-3,6-dodecadienoyl-CoA cpd: C05280; (S)-hydroxyhexanoyl-CoA cpd: C05268; (S)-hydroxyoctanoyl-CoA cpd: C05266; octanoyl-CoA cpd: C01944; (S)-hydroxydecanoyl-CoA cpd: C05264; decanoyl-CoA (n-C10: 0CoA) cpd: C05274; (S)-3-hydroxydodecanoyl-CoA cpd: C05262; lauroyl-CoA cpd: C01832; primary alcohol cpd: C00226; glutaryl-CoA cpd: C00527; (S)-3-hydroxytetradecanoyl-CoA cpd: C05260; tetradecanoyl-CoA cpd: C02593; fatty acid cpd: C00162; (S)-3-hydroxyhexadecanoyl-CoA cpd: C05258; palmitic acid cpd: C00249; (2E)-hexadecenoyl-CoA cpd: C05272; trans-2-hexenoyl-CoA cpd: C05271; crotonoyl-CoA cpd: C00877; acetoacetyl-CoA cpd: C00332; trans,cis-Lauro-2,6-dienoyl-CoA cpd: C05279; coenzyme A cpd: C00010; 3-oxohexanoyl-CoA cpd: C05269; 3-oxooctanoyl-CoA cpd: C05267; (2E)-octenoyl-CoA cpd: C05276; 3-oxodecanoyl-CoA cpd: C05265; (2E)-decenoyl-CoA cpd: C05275; 3-oxododecanoyl-CoA cpd: C05263; (2E)-dodecenoyl-CoA cpd: C03221; aldehyde cpd: C00071; 3-oxotetradecanoyl-CoA cpd: C05261; (2E)-tetradecenoyl-CoA cpd: C05273; omega-hydroxy fatty acid cpd: C03547; 3-oxohexadecanoyl-CoA cpd: C05259; L-palmitoylcarnitine cpd: C02990Tryptophan metabolism4010.58030.5443110N-acetylserotonin cpd: C00978L-tryptophan cpd: C00078; melatonin cpd: C01598; serotonin cpd: C00780; 5-hydroxykynurenamine cpd: C05638; 5-hydroxykynurenine cpd: C05651; 5-hydroxy-L-tryptophan cpd: C00643; L-formylkynurenine cpd: C02700; acetoacetyl-CoA cpd: C00332; (S)-3-hydroxybutanoyl-CoA cpd: C01144; crotonoyl-CoA cpd: C00877; glutaryl-CoA cpd: C00527; oxoadipic acid cpd: C00322; 2-amino-3-carboxymuconic acid semialdehyde cpd: C04409; 3-hydroxyanthranilic acid cpd: C00632; L-kynurenine cpd: C00328; formylanthranilic acid cpd: C05653; L-3-hydroxykynurenine cpd: C03227; 3-hydroxykynurenamine cpd: C05636; indoleacetaldehyde cpd: C00637; 5-hydroxy-N-formylkynurenine cpd: C05648; 5-hydroxyindoleacetaldehyde cpd: C05634; tryptamine cpd: C00398; indoleacrylic acid cpd: C00331; acetyl-N-formyl-5-methoxykynurenamine cpd: C05642; 6-hydroxymelatonin cpd: C05643; N-acetylserotonin cpd: C00978; formyl-5-hydroxykynurenamine cpd: C05647; 4,6-dihydroxyquinoline cpd: C05639; acetyl-CoA cpd: C00024; 2-aminomuconic acid semialdehyde cpd: C03824; 2-aminobenzoic acid cpd: C00108; L-tryptophanyl-tRNA(Trp) cpd: C03512; cinnavalininate cpd: C05640; 4-(2-amino-3-hydroxyphenyl)-2,4-dioxobutanoic acid cpd: C05645; 4-(2-aminophenyl)-2,4-dioxobutanoic acid cpd: C01252; 4,8-dihydroxyquinoline cpd: C05637; indoleacetic acid cpd: C00954; 5-hydroxyindoleacetic acid cpd: C05635; N-methylserotonin cpd: C06212; N-methyltryptamine cpd: C06213Biosynthesis of unsaturated fatty acids4210.59830.51359110Palmitic acid cpd: C00249(13Z,16Z)-docosadi-13,16-enoyl-CoA cpd: C16645; tetracosenoyl-CoA cpd: C16532; docosenoyl-CoA cpd: C16531; icosenoyl-CoA cpd: C16530; tetracosanoyl-CoA cpd: C16529; docosanoyl-CoA cpd: C16528; (7Z,10Z,13Z,16Z)-docosatetraenoyl-CoA cpd: C16170; (11Z,14Z)-icosadienoyl-CoA cpd: C16180; (7Z,10Z,13Z,16Z,19Z)-docosapentaenoyl-CoA cpd: C16166; (11Z,14Z,17Z)-icosatrienoyl-CoA cpd: C16179; palmityl-CoA cpd: C00154; stearoyl-CoA cpd: C00412; eicosanoyl-CoA cpd: C02041; oleoyl-CoA cpd: C00510; linoleoyl-CoA cpd: C02050; arachidonyl-CoA cpd: C02249; 8,11,14-eicosatrienoyl-CoA cpd: C03595; gamma-linolenoyl-CoA cpd: C03035; (4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoyl-CoA cpd: C16169; (5Z,8Z,11Z,14Z,17Z)-icosapentaenoyl-CoA cpd: C16165; alpha-linolenoyl-CoA cpd: C16162; 13,16-docosadienoic acid cpd: C16533; nervonic acid cpd: C08323; erucic acid cpd: C08316; icosenoic acid cpd: C16526; tetracosanoic acid cpd: C08320; behenic acid cpd: C08281; 7,10,13,16-docosatetraenoic acid cpd: C16527; icosadienoic acid cpd: C16525; clupanodonic acid cpd: C16513; icosatrienoic acid cpd: C16522; palmitic acid cpd: C00249; stearic acid cpd: C01530; arachidic acid cpd: C06425; oleic acid cpd: C00712; linoleic acid cpd: C01595; arachidonic acid cpd: C00219; 8,11,14-eicosatrienoic acid cpd: C03242; gamma-linolenic acid cpd: C06426; (4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoic acid cpd: C06429; eicosapentaenoic acid cpd: C06428; alpha-linolenic acid cpd: C06427Fatty acid biosynthesis4310.60710.49905110Palmitic acid cpd: C00249Tetradecanoyl-[acp] cpd: C05761; malonyl-[acyl-carrier protein] cpd: C01209; hexadecanoyl-[acp] cpd: C05764; octadecanoyl-[acyl-carrier protein] cpd: C04088; oleoyl-[acyl-carrier protein] cpd: C01203; dodecanoyl-[acyl-carrier protein] cpd: C05223; decanoyl-[acp] cpd: C05755; octanoyl-[acp] cpd: C05752; Hexanoyl-[acp] cpd: C05749; butyryl-[acp] cpd: C05745; trans-hexadec-2-enoyl-[acp] cpd: C05763; (3R)-3-hydroxypalmitoyl-[acyl-carrier protein] cpd: C04633; 3-oxohexadecanoyl-[acp] cpd: C05762; trans-tetradec-2-enoyl-[acp] cpd: C05760; (3R)-3-hydroxytetradecanoyl-[acyl-carrier protein] cpd: C04688; 3-oxotetradecanoyl-[acp] cpd: C05759; trans-dodec-2-enoyl-[acp] cpd: C05758; (R)-3-hydroxydodecanoyl-[acp] cpd: C05757; 3-oxododecanoyl-[acp] cpd: C05756; trans-Dec-2-enoyl-[acp] cpd: C05754; (3R)-3-hydroxydecanoyl-[acyl-carrier protein] cpd: C04619; 3-oxodecanoyl-[acp] cpd: C05753; trans-Oct-2-enoyl-[acp] cpd: C05751; (3R)-3-hydroxyoctanoyl-[acyl-carrier protein] cpd: C04620; 3-oxooctanoyl-[acp] cpd: C05750; trans-Hex-2-enoyl-[acp] cpd: C05748; (R)-3-hydroxyhexanoyl-[acp] cpd: C05747; 3-oxohexanoyl-[acp] cpd: C05746; but-2-enoyl-[acyl-carrier protein] cpd: C04246; (3R)-3-hydroxybutanoyl-[acyl-carrier protein] cpd: C04618; acetyl-[acyl-carrier protein] cpd: C03939; Malonyl-CoA cpd: C00083; acetyl-CoA cpd: C00024; acyl-carrier protein cpd: C00229; Holo-[carboxylase] cpd: C06250; carboxybiotin-carboxyl-carrier protein cpd: C04419; myristic acid cpd: C06424; 3-oxostearoyl-[acp] cpd: C16219; stearic acid cpd: C01530; oleic acid cpd: C00712; dodecanoic acid cpd: C02679; palmitic acid cpd: C00249; acetoacetyl-[acp] cpd: C05744Primary bile acid biosynthesis4610.63230.45839110.02976Glycine cpd: C00037Cholesterol cpd: C00187; cholest-5-ene-3beta,26-diol cpd: C15610; 25-hydroxycholesterol cpd: C15519; 7 alpha,26-dihydroxy-4-cholesten-3-one cpd: C17336; 4-cholesten-7alpha,12alpha-diol-3-one cpd: C17339; 7a-hydroxy-cholestene-3-one cpd: C05455; 5-b-cholestane-3a , 7a ,12a-triol cpd: C05454; (25R)-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-26-oyl-CoA cpd: C15613; (25S)-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-26-oyl-CoA cpd: C17343; 3a,7a,12a-trihydroxy-5b-cholest-24-enoyl-CoA cpd: C05460; 3a,7a,12a-trihydroxy-5b-24-oxocholestanoyl-CoA cpd: C05467; 3a,7a-dihydroxy-5b-24-oxocholestanoyl-CoA cpd: C05449; chenodeoxycholoyl-CoA cpd: C05337; glycine cpd: C00037; taurine cpd: C00245; 3alpha,7alpha,12alpha,26-tetrahydroxy-5beta-cholestane cpd: C05446; 3a,7a,12a-trihydroxy-5b-cholestan-26-al cpd: C01301; 3 beta,7 alpha-dihydroxy-5-cholestenoate cpd: C17335; 7-a,27-dihydroxycholesterol cpd: C06341; 7a-Hydroxycholesterol cpd: C03594; 7-a,25-dihydroxycholesterol cpd: C15520; Choloyl-CoA cpd: C01794; 24-hydroxycholesterol cpd: C13550; 3alpha,7alpha-dihydroxy-5beta-cholestanate cpd: C04554; 3a,7a-dihydroxy-5b-cholestane cpd: C05452; 3 alpha,7 alpha,26-trihydroxy-5beta-cholestane cpd: C05444; 3a,7a-dihydroxy-5b-cholestan-26-al cpd: C05445; (25R)-3alpha,7alpha-dihydroxy-5beta-cholestanoyl-CoA cpd: C17345; (25S)-3alpha,7alpha-dihydroxy-5beta-cholestanoyl-CoA cpd: C17346; 3a,7a-dihydroxy-5b-cholest-24-enoyl-CoA cpd: C05447; 3a,7a,12a-Trihydroxy-5b-cholestanoic acid cpd: C04722; 3a,7a,12a-trihydroxy-5b-cholestanoyl-CoA cpd: C05448; 3a,7a,12a,24-tetrahydroxy-5b-cholestanoyl-CoA cpd: C05450; 3 beta-hydroxy-5-cholestenoate cpd: C17333; (24S)-cholest-5-ene-3beta,7alpha,24-triol cpd: C15518; 7 alpha-hydroxy-3-oxo-4-cholestenoate cpd: C17337; 7a,12a-dihydroxy-5b-cholestan-3-one cpd: C05453; 7a-hydroxy-5b-cholestan-3-one cpd: C05451; chenodeoxycholic acid cpd: C02528; chenodeoxycholic acid glycine conjugate cpd: C05466; taurochenodesoxycholic acid cpd: C05465; 7alpha,25-dihydroxy-4-cholesten-3-one cpd: C17332; cholic acid cpd: C00695; glycocholic acid cpd: C01921; taurocholic acid cpd: C05122; 7 alpha,24-dihydroxy-4-cholesten-3-one cpd: C17331Purine metabolism6810.77490.25507110Sulfate cpd: C00059Guanosine diphosphate cpd: C00035; xanthine cpd: C00385; D-ribulose 5-phosphate cpd: C00117; phosphoribosyl pyrophosphate cpd: C00119; L-glutamine cpd: C00064; 5-phosphoribosylamine cpd: C03090; glycineamideribotide cpd: C03838; phosphoribosylformylglycineamidine cpd: C04640; AICAR cpd: C04677; SAICAR cpd: C04823; 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate cpd: C04751; RNA cpd: C00046; cyclic AMP cpd: C00575; adenosine triphosphate cpd: C00002; dATP cpd: C00131; ADP cpd: C00008; dADP cpd: C00206; adenosine monophosphate cpd: C00020; adenylsuccinic acid cpd: C03794; inosinic acid cpd: C00130; adenosine cpd: C00212; deoxyadenosine monophosphate cpd: C00360; deoxyadenosine cpd: C00559; deoxyinosine cpd: C05512; xanthosine cpd: C01762; IDP cpd: C00104; guanosine monophosphate cpd: C00144; xanthylic acid cpd: C00655; hypoxanthine cpd: C00262; inosine cpd: C00294; guanine cpd: C00242; deoxyguanosine cpd: C00330; allantoic acid cpd: C00499; uric acid cpd: C00366; 5-hydroxyisourate cpd: C11821; guanosine 3′-diphosphate 5′-triphosphate cpd: C04494; guanosine triphosphate cpd: C00044; 2′-deoxyguanosine 5′-monophosphate cpd: C00362; dGDP cpd: C00361; guanosine cpd: C00387; dGTP cpd: C00286; cyclic GMP cpd: C00942; sulfate cpd: C00059; adenosine phosphosulfate cpd: C00224; phosphoadenosine phosphosulfate cpd: C00053; 5′-Phosphoribosyl-N-formylglycinamide cpd: C04376; inosine triphosphate cpd: C00081; xanthosine 5-triphosphate cpd: C00700; diadenosine tetraphosphate cpd: C01260; P1,P4-bis(5′-xanthosyl) tetraphosphate cpd: C04392; adenosine diphosphate ribose cpd: C00301; adenine cpd: C00147; dIDP cpd: C01344; 2′-deoxyinosine triphosphate cpd: C01345; diadenosine triphosphate cpd: C06197; phosphoribosyl formamidocarboxamide cpd: C04734; 5-hydroxy-2-oxo-4-ureido-2,5-dihydro-1H-imidazole-5-carboxylate cpd: C12248; 5-aminoimidazole ribonucleotide cpd: C03373; DNA cpd: C00039; ammonia cpd: C00014; urea cpd: C00086; (S)-ureidoglycolic acid cpd: C00603; guanosine 3′,5′-bis(diphosphate) cpd: C01228; adenosine 3′,5′-diphosphate cpd: C00054; diguanosine tetraphosphate cpd: C01261; dIMP cpd: C06196; 5-amino-4-imidazolecarboxyamide cpd: C04051; (S) (+)-allantoin cpd: C02350Aminoacyl-tRNA biosynthesis6910.77990.24863110Glycine cpd: C00037L-asparagine cpd: C00152; tRNA(Asn) cpd: C01637; L-histidine cpd: C00135; tRNA(His) cpd: C01643; L-phenylalanine cpd: C00079; tRNA(Phe) cpd: C01648; L-arginine cpd: C00062; tRNA(Arg) cpd: C01636; L-glutamine cpd: C00064; tRNA(Gln) cpd: C01640; L-cysteine cpd: C00097; tRNA(Cys) cpd: C01639; glycine cpd: C00037; tRNA(Gly) cpd: C01642; tRNA(Asp) cpd: C01638; L-aspartic acid cpd: C00049; L-serine cpd: C00065; tRNA(Ser) cpd: C01650; L-methionine cpd: C00073; tRNA(Met) cpd: C01647; L-valine cpd: C00183; tRNA(Val) cpd: C01653; L-alanine cpd: C00041; tRNA(Ala) cpd: C01635; L-lysine cpd: C00047; tRNA(Lys) cpd: C01646; L-isoleucine cpd: C00407; tRNA(Ile) cpd: C01644; tRNA(Leu) cpd: C01645; L-leucine cpd: C00123; L-threonine cpd: C00188; tRNA(Thr) cpd: C01651; tRNA(Trp) cpd: C01652; L-tryptophan cpd: C00078; N10-formyl-THF cpd: C00234; L-methionyl-tRNA cpd: C02430; L-tyrosine cpd: C00082; tRNA(Tyr) cpd: C00787; L-proline cpd: C00148; tRNA(Pro) cpd: C01649; L-glutamic acid cpd: C00025; tRNA(Glu) cpd: C01641; glutaminyl-tRNA cpd: C02282; L-asparaginyl-tRNA(Asn) cpd: C03402; tRNA(Sec) cpd: C16636; L-seryl-tRNA(Sec) cpd: C06481; O-phosphoseryl-tRNA(Sec) cpd: C16638; L-histidyl-tRNA(His) cpd: C02988; L-phenylalanyl-tRNA(Phe) cpd: C03511; L-arginyl-tRNA(Arg) cpd: C02163; L-cysteinyl-tRNA(Cys) cpd: C03125; Glycyl-tRNA(Gly) cpd: C02412; L-aspartyl-tRNA(Asp) cpd: C02984; L-seryl-tRNA(Ser) cpd: C02553; L-valyl-tRNA(Val) cpd: C02554; L-alanyl-tRNA cpd: C00886; L-lysyl-tRNA cpd: C01931; L-isoleucyl-tRNA(Ile) cpd: C03127; L-leucyl-tRNA cpd: C02047; L-threonyl-tRNA(Thr)cpd: C02992; L-tryptophanyl-tRNA(Trp) cpd: C03512; tetrahydrofolic acid cpd: C00101; N-formylmethionyl-tRNA cpd: C03294; L-tyrosyl-tRNA(Tyr) cpd: C02839; L-prolyl-tRNA(Pro) cpd: C02702; L-glutamyl-tRNA(Glu) cpd: C02987; L-glutamyl-tRNA(Gln) cpd: C06112; L-aspartyl-tRNA(Asn) cpd: C06113; L-selenocysteinyl-tRNA(Sec) cpd: C06482PathwayMetabolic pathway nameTotalThe number of metabolites in this pathwayHitsThe number of differential metabolites hit this pathwayRawpP value of metabolic pathway enrichment analysis−ln (p)Minus log base E of PHolm adjustP values corrected by Holm–Bonferroni method for multiple hypothesis testingFDRP value corrected by false discovery rate (FDR) method for multiple hypothesis testingImpactImpact value of metabolic pathway topology analysisHits cpdThe names and KEGG IDs of differential metabolites hit the pathwayTotal cpdAll metabolite names and KEGG IDs contained in this pathwayFigure 3
Metabolic pathway enrichment analysis of differential metabolites. (a) Pathway analysis of group M versus group T based on mass spectrometry combined with high-performance liquid chromatography. (b) Pathway analysis of group M versus group T based on mass spectrometry combined with gas chromatography. Each bubble represents a metabolic pathway; the bubble size represents the influence factor, the larger the size, the larger the influence factor; the vertical coordinate of the bubble and the color of the bubble indicate theP value of the enrichment analysis; the darker the color, the smaller the P value, the more significant the enrichment.
(a)(b)(c)


### 3.4. Comparison of Serum Inflammatory Factor Levels in Three Groups of Mice

Finally, we observed the serum inflammatory factor levels in the three groups of mice to determine the effect of Yanghe decoction on inflammatory factors in mice with Hashimoto’s thyroiditis. The results showed that the serum inflammatory factor IL-35 was significantly lower and IL-6 was significantly higher in mice of group M than group C, while the serum inflammatory factor IL-35 was significantly higher and IL-6 was significantly lower in mice of group T than group M. The results showed that the serum inflammatory factor IL-35 was significantly lower and IL-6 was significantly lower in mice of group T than group C. See Figure4.Figure 4
Comparison of serum levels of inflammatory factors IL-35 and IL-6 in three groups of mice.


## 3.1. Principal Component Analysis

First, we performed PCA on the results of the three assays to observe the overall metabolic levels and species differences between the groups. The results of principal component analysis (positive ions) of the mass spectrometry coupled with gas and high-performance liquid phases showed that the samples of groups M and T were more closely distributed and both groups were distant from the samples of group C. The results of the NMR principal component analysis showed that the samples of groups M and T were distributed more into and partitioned from the samples of group C. This can all indicate that the more similar the type and content of metabolites in groups M and T, the more different the overall metabolic levels in groups M and T from group C. The samples were all in the 95% confidence interval. See Figure1.Figure 1
Principal component analysis. (a) Scatter plot and 3D plot of PCA scores for all samples detected by mass spectrometry coupled with the high-performance liquid phase. (b) Scatter plot and 3D plot of PCA scores for all samples detected by mass spectrometry coupled with the gas phase. (c) Scatter plot and 3D plot of PCA scores of all samples detected by NMR.
(a)(b)(c)


## 3.2. Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) and Differential Metabolite Analysis

We then modeled OPLS-DA for the M and T groups and screened for differential metabolites. The scatter plots of OPLS-DA model scores for the three assays showed that the horizontal distance between samples was farther for group M versus group T, which could indicate the greater the difference between the two groups, and the samples were very clearly differentiated. The samples were all in the 95% confidence interval. The results of the differential metabolite analysis showed that a total of 38 differential metabolites were screened for the mass spectrometry coupled with high-performance liquid phase (Table1); a total of 120 differential metabolites were screened for the mass spectrometry coupled with gas phase (Table 2), and the conditions for this metabolite screening were VIP value (the projected importance of the variable obtained from the OPLS-DA model for the comparison of the substance in this group) > 1 and P<0.05. NMR analysis showed that α-glucose and β-glucose were significant differentiating metabolites (see Figure 2).Table 1
Differential metabolites detected by mass spectrometry and high-performance liquid chromatography.
IDMS2 nameMS2 scorertmzSuperClassTypeMEAN TMEAN MVIPP-valueQ-valueFOLDCHANGELOG_FOLDCHANGE22D-Glutamine0.999291389.5485147.0759376Organic acids and derivativesForward3.7461839523.4466533471.9316642810.01476564411.0869047670.12022553947Trigonelline0.995484301.0875138.0543287Alkaloids and derivativesForward1.0249576010.8389521332.0221650480.04605110311.2217116580.28890382854N-Acetylhistamine0.994856147.787154.0968957Organic acids and derivativesForward0.0739908140.1106984541.8291806350.02558547810.668399709−0.5812169956Kanzonol K0.99465635.0689437.1927582Phenylpropanoids and polyketidesForward0.0519548910.0310480152.1012812750.00197428911.6733723930.74275853969L-Methionine0.991602301.415150.0577251Organic acids and derivativesForward0.6169196450.4680697971.9399106810.01967814411.3180078030.39835891299Ecgonine0.981134350.546186.1120848Alkaloids and derivativesForward0.0273475090.0306545131.9266898810.03169306910.892120183−0.164690017101L-histidine0.979726408.5865156.0761942Organic acids and derivativesForward0.494053370.2238941392.081970460.00858243312.2066382441.141850134107L-phenylalanine0.977905275.944166.0854692Organic acids and derivativesForward4.7637714663.8284602411.8972162090.03432289211.2443048030.315339929112L-alanine0.975801363.87990.05507238Organic acids and derivativesForward0.4379204690.3634103631.9121314980.03199470711.2050302190.269069326138Phytosphingosine0.96538157.8408318.299383Organic nitrogen compoundsforward1.7435292661.1264069121.9483855330.02199269611.5478680470.630282489141Pentanenitrile0.962912266.60484.08082795Organonitrogen compoundsForward0.2645201370.2265185882.0058009640.01882448611.1677634870.223748107153Imidazoleacetic acid riboside0.954489370.1495259.0919389Nucleosides, nucleotides, and analoguesForward0.0149655020.0194694762.0402879720.00604459310.768664826−0.3795734421563beta,6beta-Dihydroxynortropane0.94801152.1612144.1012842Alkaloids and derivativesForward0.1188181450.0864957282.0504821120.00443131111.3736880180.458054387158L-kynurenine0.944886280.281209.0914398Organic oxygen compoundsForward0.103724740.0828028411.9877219230.01317307711.2526712630.32500786160Arecaidine0.944798282.167142.0858524Alkaloids and derivativesForward0.0800852940.0530141312.0167966560.02200487211.5106405180.595160389161Carnosine0.942805434.3945227.113435Organic acids and derivativesForward0.0480449360.0663721361.8190562390.03111277110.723872074−0.466193334169Thelephoric acid0.935613407.237353.0278188Organoheterocyclic compoundsForward0.0026053810.0037754051.8061407080.03321201710.690093214−0.535136849180L-acetylcarnitine0.92253320.788204.1225337Lipids and lipid-like moleculesForward3.6985298422.8399080031.8771470180.02489894611.3023414270.381107721204Alpha-methylstyrene0.89864235.2889119.0850948BenzenoidsForward0.1884414730.1316320932.1598874960.00157772911.4315769780.517605248228Glycerophosphocholine0.86945400.206258.1089005Lipids and lipid-like moleculesForward0.6159519630.9257415111.8646435320.03582193310.665360638−0.5877915732301-(beta-D-ribofuranosyl)-1,4-dihydronicotinamide0.865109171.668257.1128126Organic oxygen compoundsForward0.1173256080.1385665812.1380359110.00478950910.846709269−0.240061413238D-biopterin0.853104272.631238.0928999Organoheterocyclic compoundsForward0.0264860360.0214462491.828256740.04043988811.2349962320.30450664240N-(3-methylbutyl)acetamide0.850798268.9475130.1221116Organic acids and derivativesForward0.0582095810.0497433652.1420982630.00062013611.1701979120.226752549254N-acetylornithine0.829892374.192175.1070165Organic acids and derivativesForward0.0562798930.0470083061.8305870280.03537067211.1972329430.2597038822591-(Hydroxymethyl)-5,5-dimethyl-2,4-imidazolidinedione0.818992407.131159.0757679Organoheterocyclic compoundsForward0.1820869830.155835561.9456649270.01444207511.1684559280.224603319291Biochanin A0.76976639.59165285.07362Phenylpropanoids and polyketidesforward0.0317217030.0417325311.9202547120.01825747810.760119332−0.395702167295Heptadecanoyl carnitine0.756939194.733414.3571643Lipids and lipid-like moleculesForward0.0174031640.0221186512.1376965730.0007222710.786809469−0.3459137753153-[(3-Methylbutyl)nitrosoamino]-2-butanone0.71523136.73805187.1434871Organooxygen compoundsForward0.3399653070.4571208562.0613000250.00897733110.743709903−0.4271881123202-Acetylpyrazine0.710279330.908123.0549956Organooxygen compoundsForward0.0131233240.0087841521.9704703890.0202078611.4939773690.579158294328Sedoheptulose 1-phosphate0.70085440.022291.0477486Organic oxygen compoundsForward0.0510976910.0693458062.0465084060.00706959110.736853377−0.440550522360Glechomafuran0.646208224.3045231.1407859Lipids and lipid-like moleculesForward0.0100339710.0147501962.0959910590.00284462710.680260196−0.55584142384LysoPC(17 : 0)0.611195214.164510.3549507Lipids and lipid-like moleculesForward0.7720316391.0468688231.8899345340.04551365810.73746741−0.4393488410Acetyl-N-formyl-5-methoxykynurenamine0.57791624.7427265.1210007Organic oxygen compoundsForward0.0104525780.0064759942.189496149.71037E−0511.6140499980.690685269430Cysteinyl-serine0.56069125.0757209.0581161Organic acids and derivativesForward0.5428060050.2856816991.9417437440.04101055411.9000377260.926028064435L-octanoylcarnitine0.556023224.21288.2159451Lipids and lipid-like moleculesForward0.0644109740.0896772522.1069708410.00228456510.718253205−0.47743557442S-methylmethionine0.553207336.697164.0731609Forward0.0325733180.0448692741.8469457230.03290781810.725960445−0.4620371524714-Aminohippuric acid0.488799315.061184.0895643BenzenoidsForward0.0093285130.0230728761.9116708970.01025103610.404306452−1.306478867477PE(16 : 0/18 : 3(9Z,12Z,15Z))0.471196164.6285714.5059277Lipids and lipid-like moleculesForward0.0431752760.027338731.8381578390.03266264211.5792714560.659259173IDThe unique data number of the substance in this qualitative analysisMS2 nameThe substance name obtained by qualitative matching analysis of secondary mass spectrometryMS2 scoreThe score of the second-level match, with the value [0, 1]rtChromatographic retention time of the substancemzMaterial characteristics mass charge ratio of ionsSuperClassClassification information of the substance in the HMDB databaseTypeThe matching mode includes MS2 forward/reverse matching (MS2 forward/reverse), first-order average molecular weight/single isotope molecular weight matching and no matchingMEAN OOThe mean of the relative quantitative value of the substance in one of the experimental groups within the group comparisonMEAN XXThe mean of the relative quantitative value of the substance in the other experimental group within the group comparisonVIPThe substance in the group was compared to the OPLS-DA model obtained by the variable projection significanceP-valueThep-value obtained by the t-test of this substance in the group comparison, p-value = probabilityof hypothesis being correct but rejected = number of negative results/total number of results, is a test probability against the sample dataQ-valueThe hypothesis test statistic (p-value) is the result after the correction of multiple hypothesis test. Q value = the probability of being rejected but correct = the number of false-positive results/thenumber of presumed positive results. It is a test probability of the inference obtained from statistical test and the re-statistics of p-valueFOLD CHANGEThe substance in this group was compared with the multiple relationship between the two groups of experimentsLOG_FOLDCHANGEFOLD CHANGE takes the logarithm base 2Table 2
Differential metabolites detected by mass spectrometry combined with gas phase.
IDPeak2SimilarityrtCountMassMEAN TMEAN MVIPP-valueQ-valueFOLDCHANGELOG_FOLDCHANGE692-Hydroxybutanoic acid9235.90393,091311.2725150661.8291571.2998433990.0308247660.0547177460.695683796−0.523496376873-Hydroxybutyric acid9176.15207,0914725.4876259738.15091.4357728810.0069065930.0310583520.668074026−0.581920125228L-malic acid9108.38126,097315.0209830424.188151.2068502040.0458445990.0647071690.621005853−0.68732123407Palmitoleic acid90311.4327,09554.4277154536.2580291.2366459830.0496532090.0670783250.707525543−0.499145861410Palmitic acid87811.5306,0911720.9099871628.737531.3098507060.0250560750.050260230.727619379−0.45874412768Glycine 18765.88319,091023.9205308895.9694691.4371491150.0036231460.0310583520.656763806−0.606553473144glycerol8556.94956,0920514.7360911322.119351.3165077760.0303746470.0544071640.66620819−0.585955005234Threitol8528.47978,092170.3205982890.5348071.4397500270.0032849070.0310583520.599465913−0.738250374273Alpha-ketoglutaric acid8268.93667,09734.3927607567.0408611.190463660.049580740.0670351060.623895417−0.680623882172Uracil8227.43667,092410.3821254020.6614691.3530534570.0220607250.0474554370.577691894−0.791627843184Pelargonic acid7947.60111,092150.0463908640.075661.232558620.0438261990.0633623510.613151594−0.705684289222Aminomalonic acid7918.28837,092180.4134769310.5848891.2444331470.0313880310.0550986310.706932665−0.500355292062-Deoxytetronic acid7808.01104,09734.2877756646.9928051.3806595290.0077585490.0310583520.613169609−0.705641901853-Hydroxypyridine7786.0817,091520.2330751930.3495791.4734374490.0041902230.0310583520.666731544−0.5848221111274-Hydroxybutyrate7696.69919,092330.0183865990.0323411.340645620.0251107420.0503079060.568527482−0.814698006427heptadecanoic acid74511.985,091170.1105081210.1685021.3367032070.0320822580.0555565870.655828223−0.608610108197Thymine6777.85356,092700.0437783810.0661041.3716052110.0129065660.0378012160.662263926−0.59452182110Malonic acid 16746.46348,091410.1201531860.2125151.4809746110.0015763690.0310583520.56538798−0.82268688399Methyl phosphate6656.28585,092410.1156624740.2116871.4639365010.0027270240.0310583520.5463833−0.872014707207Beta-alanine 26438.03652,092480.0540565270.1013641.4210354910.0149506270.0396599960.533289189−0.9070100131622,3-Dihydroxypyridine6337.28778,092400.0864322910.129721.3933943510.0066638440.0310583520.666297891−0.585760767463N-acetyl-5-hydroxytryptamine 159013.2097,09290160.0062516231.71931.3030210850.0414787310.0617143610.690517501−0.5342501151133-Aminoisobutyric acid 25206.51726,09570.322001070.4964841.3389674690.0156377140.040208520.648562563−0.6246823453071,2-Cyclohexanedione 45179.492,082050.1017488130.1816781.3875714470.0266165590.0515765050.560051285−0.836369152216Erythrose 15148.18007,093500.6899316791.0920361.2724094680.0283014720.0529008680.631784782−0.662494907219Capric acid5088.24689,091170.1612120230.2745971.3585432350.0107270660.0353398220.587086555−0.7683548781462-Deoxyuridine5037.01356,03810.0002580020.6598531.5390405050.0161230390.0405760960.000390999−11.32054852377D-altrose 248710.8282,032570.0002580020.1383831.5418037310.0002791560.0310583520.0018644−9.067072758111Methylmalonic acid4816.46941,092260.2104692690.4239131.4962354510.0022031880.0310583520.496491996−1.010157634285Digitoxose 14529.08156,03730.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.589515221246trans-4-Hydroxy-L-proline 24498.64244,031860.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.58951522188Sulfuric acid4196.16896,092810.7595822161.1403431.3211894910.0300787260.0541998950.666099824−0.5861896952182,4-Diaminobutyric acid 34168.2337,091030.1773469780.2799411.3706080870.0091077190.0330745620.633516577−0.658545725425d-Glucoheptose 141211.9031,073190.0122581460.0210771.3154295160.0393163990.0603777040.581574962−0.781962934501Isomaltose 241015.118,07730.216801480.3308111.4627086030.0053479210.0310583520.655363457−0.609632863211N-acetyl-L-leucine 24068.12433,08860.0586787620.0871621.4475791250.0111256310.0358348260.673218373−0.570853545309L-homoserine 33969.52289,062900.0521601150.0758241.4817961590.002094980.0310583520.687909761−0.539708769772-Ketovaleric acid 23935.97089,09890.1480174450.2483581.447203690.0043150570.0310583520.595983832−0.7466549022384-Acetamidobutyric acid 23878.55919,091740.2626814940.4144911.3043952180.0239233440.0492452540.633744092−0.658027703343Biuret 238510.0571,061710.1311124380.111931.2984911140.0387837810.0600351661.1713794590.228208501149O-acetylserine 23847.08467,031160.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.589515221170Oxamide3827.37756,091160.6380630280.9637431.3750597840.0109468650.0356155660.66206751−0.594949762193Resorcinol3647.74837,092400.18937030.2705611.4221353430.0288400310.0533046360.699917794−0.514742608342-Ketobutyric acid 23595.37696,091743.8953987136.3109691.4527537710.0038196660.0310583520.617242587−0.69609049495Maltose34414.4673,032040.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.589515221506Ergosterol33917.6037,071290.0381424770.1700621.1520560640.0404762470.0611051750.224285234−2.156593449290D-erythronolactone 23389.18748,092440.2335205670.3354041.3387417370.0152381130.039893680.696237385−0.522348811417Sinapyl alcohol 133111.7691,032410.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.589515221416N-acetyl-D-galactosamine 132911.7478,032020.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.5895152211143-Methylamino-1,2-propanediol 13276.52733,091160.1459287130.2519511.4877100760.0005752240.0310583520.579194579−0.787879996924-Hydroxypyridine3266.20052,091410.4899277930.7959271.4513239710.0024659510.0310583520.615543839−0.700066486251Cytosine3228.68941,093142.5098405524.0820561.2831599780.0219633170.0473573920.614847194−0.701700187507Cholestane-3,5,6-triol, (3?5?6?-32118.7661,09570.8851466532.0021861.3875648920.0069055780.0310583520.442090187−1.1775873842952-Carboxybenzaldehyde3189.29889,034480.0002580020.0405661.541941440.0018885660.0310583520.006360115−7.296731326486Alizarin29613.9349,031920.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.589515221255Maleamate 22728.72638,072440.0179845420.0832381.1339189220.0230974270.0484710180.216061427−2.2104865641694-Acetylbutyric acid 22657.37059,09710.8804394141.4448541.4367706410.0187648950.0438594070.60936233−0.7146277771Analyte 104.85415,092200.1436275880.2069941.4125125280.0093754160.033479910.693874841−0.5272526372Analyte 204.86659,092210.9391797891.4825621.2231874220.0485398460.0664064660.633484237−0.6586193734Analyte 404.906,0912725.0483889140.416221.4272777130.0240226420.0493363510.619760853−0.6902164649Analyte 904.95133,09560.0760087240.1305491.3871058780.0064814080.0310583520.582225673−0.78034963913Analyte 1305.01281,092810.5191585430.881591.4159562770.004327660.0310583520.588888971−0.76393244114Analyte 1505.05415,09890.1266863660.1943851.3707323850.0113142690.0360614850.651727984−0.61765815315Analyte 1605.06096,09710.142475830.2545961.3824156910.0251283130.0503232040.559614273−0.83749533616Analyte 1705.10467,092480.1892362390.2774121.1858621230.0463494550.0650336210.682149873−0.55183935117Analyte 1805.1217,09710.235375970.3690891.2852373470.0282230690.0528413250.63772183−0.64900082821Analyte 2205.17593,091600.1173364380.2273761.3967641480.0147131460.0394622340.516045471−0.95442990226Analyte 2705.21607,092071.5341230142.4423951.4067900330.0051169320.0310583520.628122392−0.67088239428Analyte 2905.28378,093180.4905830820.816121.4367950450.0033191470.0310583520.601116114−0.734284429Analyte 3005.28985,091410.4241924950.6995251.4238410190.0233623250.0487226040.606400455−0.7216572631Analyte 3205.306,09820.1138901230.1580431.3007670640.0410701670.0614682230.720626315−0.47267675935Analyte 3605.38238,072650.0966598410.1409971.3235885580.0263448320.0513537720.685547745−0.54467094942Analyte 4305.50956,063180.0531479620.1166361.3890437740.0049726260.0310583520.45567417−1.13392550243Analyte 4405.53119,09831.0926466831.8996171.3748646830.0144040960.0391982180.575193161−0.79788157245Analyte 4605.57133,091730.0406811950.0619561.2805314430.0325395280.0558515340.656616292−0.60687754753Analyte 5405.69844,03720.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.58951522160Analyte 6105.77652,091870.4117390970.6407321.3818764880.0342612640.0569169130.642607665−0.63798990661Analyte 6205.78067,09890.1962076650.2807791.2497140740.0324071580.0557666890.698796716−0.51705526765Analyte 6605.85193,092581.1027398691.8985711.459403240.0028411140.0310583520.58082638−0.78382111666Analyte 6705.86748,092450.751411871.0420031.3408555380.0147209080.0394687670.721122764−0.47168320970Analyte 7105.91167,08830.1246001320.210661.4601880.0059234320.0310583520.5914747−0.75761163671Analyte 7205.917,082060.1098841710.152681.265734030.0412871140.0615992840.719702949−0.47452652679Analyte 8005.97948,091300.065456180.1038261.4575718180.0026013190.0310583520.630443919−0.66556005283Analyte 8406.06822,09890.6298836150.8571531.3489649080.021462270.0468456660.734855621−0.44446726686Analyte 8706.1057,093550.0131006010.0233431.4648594920.0029202620.0310583520.561219926−0.83336186394Analyte 9506.23356,093220.0910504220.1412991.3359358170.0130503720.0379445490.644383212−0.63400918896Analyte 9706.25607,092210.2733344860.5051441.3827182810.0184611580.0434964260.541101922−0.8860277397Analyte 9806.26304,092670.0521838010.0813531.3390446480.0218878510.0472811140.641446738−0.64059861798Analyte 9906.28007,091521.2432812841.7312471.3624503450.0181108880.043070520.718142143−0.477658667100Analyte 10106.35356,092110.1499751120.2432341.2694344250.0344425560.0570251210.6165884−0.697620347101Analyte 10206.37207,092210.1260686150.2297191.3698120920.0149189150.0396338390.548793895−0.865663663112Analyte 11406.51,032070.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.589515221124Analyte 12606.667,041590.0059801180.0353981.2067438520.0075958190.0310583520.168941731−2.565402355132Analyte 13406.76096,091020.0871561260.131351.4244133830.0046944750.0310583520.663543315−0.591737451135Analyte 13706.83089,091080.1974753330.3222811.3940145940.0094837190.0336400960.612743258−0.70664539138Analyte 14006.86778,093191.8731576393.1141221.4183622990.0078473030.0310583520.601504164−0.73335337147Analyte 14907.02956,09299230.5117654328.73471.3685011320.0144506120.0392384470.70120909−0.512083397150Analyte 15207.08911,031080.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.589515221168Analyte 17007.36319,092210.0386756130.0643061.4252992040.0037797370.0310583520.601426746−0.733539068174Analyte 17607.44822,031740.0002580020.0003881.3822977170.0078984880.0310583520.664566179−0.589515221176Analyte 17807.4663,093000.140669290.254311.3977994840.0093902640.0335019990.553141309−0.854280007183Analyte 18507.57178,092410.0346598520.053531.2789094920.0226391210.0480282390.647485316−0.62708062188Analyte 19007.65457,071520.0071840550.0273391.1175067830.0480282230.0660919740.262776377−1.92809251210Analyte 21208.07785,093270.0580094660.1015891.3813210630.0087969010.0325863550.571020535−0.808385465213Analyte 21508.15637,093020.0642571010.0987221.2462777130.0280986540.0527464330.650889175−0.619516174215Analyte 21708.16556,092810.0393451220.0804911.3749052610.0036862170.0310583520.48881664−1.032634697227Analyte 22908.36156,093410.0524487590.0869461.3252188160.0170928860.0417861740.603232561−0.72921379229Analyte 23108.40367,081000.2031800310.329391.3419799580.0136931480.0385605360.616837794−0.697036932237Analyte 24008.54081,093370.250365220.4556551.3667772460.0368323950.0587320650.549461995−0.863908397253Analyte 25608.70644,092920.3575289290.5159311.219381210.0484530830.0663533910.692978537−0.529117425256Analyte 25908.74333,082450.0488457080.0908291.3266294340.0097239220.0339878290.537777275−0.894919303259Analyte 26208.78319,092630.130882510.2310691.3448605660.0444344420.0637744450.566422421−0.820049721267Analyte 27008.88467,09710.112272970.2041381.4330632660.006086670.0310583520.549984911−0.862536055345Analyte 350010.0673,052920.0002580020.0637221.5396282120.0049526840.0310583520.004048846−7.948273624364Analyte 369010.4758,03550.0002580020.0635521.5422249230.0162541120.0406727060.004059669−7.944422022393Analyte 400011.1802,062170.0617136490.1615871.4056551090.0135228450.0384011380.38192255−1.38864799435Analyte 442012.219,04670.0056926750.0304991.1844972070.0394247710.0604467360.186653558−2.421565088455Analyte 463012.8955,092323.4423798154.6092311.3595474290.0253858680.0505460710.746844736−0.421119747487Analyte 495013.9389,062590.0136908520.0270191.4015705030.0231381990.0485099450.506703381−0.98078664503Analyte 511015.8464,093570.0805725970.1451651.2708592360.0356178610.0578806230.555041297−0.849332977IDThe unique data number of the substance in this qualitative analysisPeakThe name of the substance obtained by qualitative analysisSimilarityThe matching degree between the substance and the substance in the standard library in qualitative analysis, and the value is [0, 1000]rtChromatographic retention time of the substanceCountThe number of times the substance was detected in all experimental groupsMassMaterial characteristics Mass charge ratio of ionsMEAN OOThe mean of the relative quantitative value of the substance in one of the experimental groups within the group comparisonMEAN XXThe mean of the relative quantitative value of the substance in the other experimental group within the group comparisonVIPThe substance in the group was compared to the OPLS-DAmodel obtained by the variable projection significanceP-valueThep-value obtained by the t-test of this substance in the group comparison, p-value = probability of hypothesis being correct but rejected = number of negative results/total number of results, is a test probability against the sample dataQ-valueThe hypothesis test statistic (p-value) is the result after the correction of multiple hypothesis test. Q-value = the probability of being rejected but correct = the number of false-positive results/the number of presumed positive results. It is a test probability of the inference obtained from statistical test and the re-statistics of p-valueFOLD CHANGEThe substance in this group was compared with the multiple relationship between the two groups of experimentsLOG_FOLDCHANGEFOLD CHANGE takes the logarithm base 2Figure 2
OPLS-DA model score scatter plot and differential metabolite analysis. (a) Scatter plot of OPLS-DA model score and volcano plot of differential metabolites for group M versus group T detected by mass spectrometry coupled with the high-performance liquid phase. (b) Scatter plot of OPLS-DA model score and volcano plot of differential metabolites for group M versus group T detected by mass spectrometry coupled with gas phase. The size of scattering in the volcano plot represents the VIP value of OPLS-DA model, the larger the scatter the larger the VIP value. Significantly upregulated metabolites are shown in red, significantly down-regulated metabolites are shown in blue, and nonsignificantly different metabolites are shown in gray. (c) Scatter plot of OPLS-DA model scores for group M versus group T detected by NMR. (d) OPLS-DA loadings plot for group M versus group (T). The horizontal coordinates in the plot indicate the magnitude of chemical shifts, from right to left, with increasing values of chemical shifts; the vertical coordinates indicate the magnitude of loadings after back conversion, where the shades of color indicate the magnitude of correlation coefficients, red indicates a positive correlation, and blue indicates negative correlation. The marked substances in the figure are the differential metabolites that passed the critical value test of the correlation coefficient.
(a)(b)(c)(d)


## 3.3. Enrichment Analysis of Metabolic Pathways of Differential Metabolites

We further analyzed the effect of differential metabolites on their pathways. The analysis based on the mass spectrometry combined with high-performance liquid phase detection results showed that the differential metabolites in the M and T groups were enriched to 11 pathways (Table3), among which the pathways with higher enrichment were histidine metabolism, amino acid tRNA biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, tryptophan metabolism, phenylalanine metabolism, cysteine, and methionine metabolism. The analysis based on the results of mass spectrometry combined with gas-phase detection showed that the differential metabolites of the M and T groups were enriched to 28 pathways (Table 4), among which the pathways with higher enrichment were pyrimidine metabolism, biosynthesis of pantothenic acid and CoA, metabolism of β-alanine, sulfur metabolism, glycerolipid metabolism, and metabolism of glycine, serine, and threonine. Subsequently, we took the intersection of the pathways enriched by the differential metabolites in the M and T groups for both high-performance liquid- and gas-phase methods coupled to mass spectrometry and obtained a total of five common pathways (biosynthesis of amino acid tRNA, metabolism of D-glutamine and D-glutamate, metabolism of tryptophan, metabolism of nitrogen, and metabolism of arginine and proline). See Figure 3Table 3
Enrichment analysis results based on mass spectrometry combined with HPLC.
PathwayTotalHitsRawp−ln (p)Holm adjustFDRImpactHits cpdTotal cpdHistidinemetabolism1520.0115264.46320.950.945120.24194L-Histidine cpd:C00135; carnosine cpd: C00386L-glutamic acid cpd: C00025; 4-imidazolone-5-propionic acid cpd: C03680; urocanic acid cpd: C00785; L-histidine cpd: C00135; imidazole-4-acetaldehyde cpd: C05130; 1-methylhistamine cpd: C05127; methylimidazole acetaldehyde cpd: C05827; histamine cpd: C00388; carnosine cpd: C00386; N-formyl-L-aspartate cpd: C01044; formiminoglutamic acid cpd: C00439; imidazoleacetic acid cpd: C02835; methylimidazoleacetic acid cpd: C05828; L-aspartic acid cpd: C00049; 1-methylhistidine cpd: C01152Aminoacyl-tRNA biosynthesis6930.0392323.2383110L-histidine cpd: C00135; L-phenylalanine cpd: C00079; L-methionine cpd: C00073L-asparagine cpd: C00152; tRNA(Asn) cpd: C01637; L-histidine cpd: C00135; tRNA(His) cpd: C01643; L-phenylalanine cpd: C00079; tRNA(Phe) cpd: C01648; L-arginine cpd: C00062; tRNA(Arg) cpd: C01636; L-glutamine cpd: C00064; tRNA(Gln) cpd: C01640; L-cysteine cpd: C00097; tRNA(Cys) cpd: C01639; glycine cpd: C00037; tRNA(Gly) cpd: C01642; tRNA(Asp) cpd: C01638; L-aspartic acid cpd: C00049; L-serine cpd: C00065; tRNA(Ser) cpd: C01650; L-methionine cpd: C00073; tRNA(Met) cpd: C01647; L-valine cpd: C00183; tRNA(Val) cpd: C01653; L-alanine cpd: C00041; tRNA(Ala) cpd: C01635; L-lysine cpd: C00047; tRNA(Lys) cpd: C01646; L-isoleucine cpd: C00407; tRNA(Ile) cpd: C01644; tRNA(Leu) cpd: C01645; L-leucine cpd: C00123; L-threonine cpd: C00188; tRNA(Thr) cpd: C01651; tRNA(Trp) cpd: C01652; L-tryptophan cpd: C00078; N10-formyl-thf cpd: C00234; L-methionyl-tRNA cpd: C02430; L-tyrosine cpd: C00082; tRNA(Tyr) cpd: C00787; L-proline cpd: C00148; tRNA(Pro) cpd: C01649; L-glutamic acid cpd: C00025; tRNA(Glu) cpd: C01641; glutaminyl-tRNA cpd: C02282; L-asparaginyl-tRNA(Asn) cpd: C03402; tRNA(Sec) cpd: C16636; L-seryl-tRNA(Sec) cpd: C06481; O-phosphoseryl-tRNA(Sec) cpd: C16638; L-histidyl-tRNA(His) cpd: C02988; L-phenylalanyl-tRNA(Phe) cpd: C03511; L-arginyl-tRNA(Arg) cpd: C02163; L-cysteinyl-tRNA(Cys) cpd: C03125; glycyl-tRNA(Gly) cpd: C02412; L-aspartyl-tRNA(Asp) cpd: C02984; L-seryl-tRNA(Ser) cpd: C02553; L-valyl-tRNA(Val) cpd: C02554; L-alanyl-tRNA cpd: C00886; L-lysyl-tRNA cpd: C01931; L-isoleucyl-tRNA(Ile) cpd: C03127; L-leucyl-tRNA cpd: C02047; L-threonyl-tRNA(Thr) cpd: C02992; L-tryptophanyl-tRNA(Trp) cpd: C03512; tetrahydrofolic acid cpd: C00101; N-formylmethionyl-tRNA cpd: C03294; L-tyrosyl-tRNA(Tyr) cpd: C02839; L-prolyl-tRNA(Pro) cpd: C02702; L-glutamyl-tRNA(Glu) cpd: C02987; L-glutamyl-tRNA(Gln) cpd: C06112; L-aspartyl-tRNA(Asn) cpd: C06113; L-selenocysteinyl-tRNA(Sec) cpd: C06482Phenylalanine, tyrosine, and tryptophan biosynthesis410.0444533.1133110.5L-phenylalanine cpd: C00079Phenylpyruvic acid cpd: C00166; L-phenylalanine cpd: C00079; L-tyrosine cpd: C00082; 4-hydroxyphenylpyruvic acid cpd: C01179D-glutamine and D-glutamate metabolism510.0552732.8955110D-glutamine cpd: C00819L-glutamic acid cpd: C00025; D-glutamine cpd: C00819; L-glutamine cpd: C00064; oxoglutaric acid cpd: C00026; D-glutamic acid cpd: C00217Tryptophan metabolism4020.0726662.6219110.10987L-kynurenine cpd: C00328; acetyl-N-formyl-5-methoxykynurenamine cpd: C05642L-tryptophan cpd: C00078; melatonin cpd: C01598; serotonin cpd: C00780; 5-hydroxykynurenamine cpd: C05638; 5-hydroxykynurenine cpd: C05651; 5-hydroxy-L-tryptophan cpd: C00643; L-formylkynurenine cpd: C02700; acetoacetyl-CoA cpd: C00332; (S)-3-hydroxybutanoyl-CoA cpd: C01144; crotonoyl-CoA cpd: C00877; glutaryl-CoA cpd: C00527; oxoadipic acid cpd: C00322; 2-amino-3-carboxymuconic acid semialdehyde cpd: C04409; 3-hydroxyanthranilic acid cpd: C00632; L-kynurenine cpd: C00328; formylanthranilic acid cpd: C05653; L-3-hydroxykynurenine cpd: C03227; 3-hydroxykynurenamine cpd: C05636; indoleacetaldehyde cpd: C00637; 5-hydroxy-N-formylkynurenine cpd: C05648; 5-hydroxyindoleacetaldehyde cpd: C05634; tryptamine cpd: C00398; indoleacrylic acid cpd: C00331; acetyl-N-formyl-5-methoxykynurenamine cpd: C05642; 6-hydroxymelatonin cpd: C05643; N-acetylserotonin cpd: C00978; formyl-5-hydroxykynurenamine cpd: C05647; 4,6-dihydroxyquinoline cpd: C05639; acetyl-CoA cpd: C00024; 2-aminomuconic acid semialdehyde cpd: C03824; 2-aminobenzoic acid cpd: C00108; L-tryptophanyl-tRNA(Trp) cpd: C03512; cinnavalininate cpd: C05640; 4-(2-amino-3-hydroxyphenyl)-2,4-dioxobutanoic acid cpd: C05645; 4-(2-aminophenyl)-2,4-dioxobutanoic acid cpd: C01252; 4,8-dihydroxyquinoline cpd: C05637; indoleacetic acid cpd: C00954; 5-hydroxyindoleacetic acid cpd: C05635; N-methylserotonin cpd: C06212; N-methyltryptamine cpd: C06213Nitrogen metabolism910.0974152.3288110L-histidine cpd: C00135Ammonia cpd: C00014; carbon dioxide cpd: C00011; L-glutamic acid cpd: C00025; L-Glutamine cpd: C00064; L-cystathionine cpd: C02291; L-Histidine cpd: C00135; carbamoylphosphate cpd: C00169; carbonic acid cpd: C01353; glycine cpd: C00037Phenylalanine metabolism1110.117822.1386110.40741L-phenylalanine cpd: C00079Phenylacetaldehyde cpd: C00601; phenylacetyl-CoA cpd: C00582; L-phenylalanine cpd: C00079; phenylethylamine cpd: C05332; phenylpyruvic acid cpd: C00166; phenylacetic acid cpd: C07086; phenylacetylglycine cpd: C05598; ortho-hydroxyphenylacetic acid cpd: C05852; enol-phenylpyruvate cpd: C02763; 2-phenylacetamide cpd: C02505; L-tyrosine cpd: C00082Sphingolipid metabolism2110.213511.5441110Phytosphingosine cpd: C12144Sphinganine cpd: C00836; ceramide 1-phosphate cpd: C02960; sphingosine 1-phosphate cpd: C06124; sphinganine 1-phosphate cpd: C01120; dihydroceramide cpd: C12126; phytoceramide cpd: C12145; SM cpd: C00550; ceramide cpd: C00195; Palmityl-CoA cpd: C00154; L-Serine cpd: C00065; 3-dehydrosphinganine cpd: C02934; galabiosylceramide cpd: C06126; galactosylceramide cpd: C02686; GM4 cpd: C06128; lactosylceramide cpd: C01290; glucosylceramide cpd: C01190; sphingosine cpd: C00319; 3-O-Sulfogalactosylceramide (d18 :  : 1/24 :  : 0) cpd: C06125; phytosphingosine cpd: C12144; digalactosylceramidesulfate cpd: C06127; O-phosphoethanolamine cpd: C00346Cysteine and methionine metabolism2710.266161.3236110.08685L-methionine cpd: C000732-Oxo-4-methylthiobutanoic acid cpd: C01180; 1,2-dihydroxy-3-keto-5-methylthiopentene cpd: C15606; 5′-methylthioadenosine cpd: C00170; S-adenosylmethioninamine cpd: C01137; S-adenosylmethionine cpd: C00019; L-cystathionine cpd: C02291; L-homocysteine cpd: C00155; L-serine cpd: C00065; L-methionine cpd: C00073; S-adenosylhomocysteine cpd: C00021; 2,3-diketo-5-methylthiopentyl-1-phosphate cpd: C15650; cysteic acid cpd: C00506; L-cystine cpd: C00491; L-cysteine cpd: C00097; 3-sulfinoalanine cpd: C00606; sulfite cpd: C00094; 3-mercaptopyruvic acid cpd: C00957; 3-methylthiopropionic acid cpd: C08276; 5-methylthioribose 1-phosphate cpd: C04188; 2-ketobutyric acid cpd: C00109; 2-aminoacrylic acid cpd: C02218; 3-sulfopyruvic acid cpd: C05528; thiocysteine cpd: C01962; 3-sulfinylpyruvic acid cpd: C05527; pyruvic acid cpd: C00022; thiosulfate cpd: C00320; 3-mercaptolactic acid cpd: C05823Glycerophospholipid metabolism3010.291231.2336110.02315Glycerophosphocholine cpd: C00670Phosphatidylethanolamine cpd: C00350; phosphatidylcholine cpd: C00157; dihydroxyacetone phosphate cpd: C00111; LysoPC (18 : 1(9Z)) cpd: C04230; 1,2-diacyl-sn-glycerol cpd: C00641; citicoline cpd: C00307; phosphorylcholine cpd: C00588; choline cpd: C00114; acetylcholine cpd: C01996; O-phosphoethanolamine cpd: C00346; ethanolamine cpd: C00189; PA (16 :  : 0/16 :  : 0) cpd: C00416; acyl-CoA cpd: C00040; 1-acyl-sn-glycerol 3-phosphate cpd: C00681; CDP-diacylglycerol cpd: C00269; glycerol 3-phosphate cpd: C00093; 1-acyl-sn-glycero-3-phosphoethanolamine cpd: C04438; 2-acyl-sn-glycero-3-phosphoethanolamine cpd: C05973; 2-acyl-sn-glycero-3-phosphocholine cpd: C04233; CDP-glycerol cpd: C00513; PS (16 : 0/16 : 0) cpd: C02737; phosphatidylglycerol cpd: C00344; dihydroxyacetone phosphate acyl ester cpd: C03372; CDP-ethanolamine cpd: C00570; 1-phosphatidyl-D-myo-inositol cpd: C01194; glycerylphosphorylethanolamine cpd: C01233; glycerophosphocholine cpd: C00670; phosphatidyl-N-methylethanolamine cpd: C01241; phosphatidylglycerophosphate cpd: C03892; cardiolipin cpd: C05980Arginine and proline metabolism4410.397960.9214110N-acetylornithine cpd: C00437L-glutamine cpd: C00064; ammonia cpd: C00014; carbamoylphosphate cpd: C00169; ornithine cpd: C00077; citrulline cpd: C00327; L-aspartic acid cpd: C00049; argininosuccinic acid cpd: C03406; L-arginine cpd: C00062; L-glutamic acid cpd: C00025; N-acetylornithine cpd: C00437; L-proline cpd: C00148; peptide cpd: C00012; D-proline cpd: C00763; hydroxyproline cpd: C01157; pyrroline hydroxycarboxylic acid cpd: C04281; L-4-hydroxyglutamate semialdehyde cpd: C05938; L-erythro-4-hydroxyglutamate cpd: C05947; N-(o)-hydroxyarginine cpd: C05933; guanidinoacetic acid cpd: C00581; creatine cpd: C00300; gamma-aminobutyric acid cpd: C00334; agmatine cpd: C00179; L-glutamic-gamma-semialdehyde cpd: C01165; L-glutamic acid 5-phosphate cpd: C03287; (S)-1-pyrroline-5-carboxylate cpd: C03912; putrescine cpd: C00134; 4-aminobutyraldehyde cpd: C00555; S-adenosylmethioninamine cpd: C01137; S-adenosylmethionine cpd: C00019; spermidine cpd: C00315; N-acetylputrescine cpd: C02714; N4-acetylaminobutanal cpd: C05936; cis-4-hydroxy-D-proline cpd: C03440; fumaric acid cpd: C00122; urea cpd: C00086; N-acetyl-L-alanine cpd: C00624; 1-pyrroline-2-carboxylic acid cpd: C03564; D-4-hydroxy-2-oxoglutarate cpd: C05946; nitric oxide cpd: C00533; phosphocreatine cpd: C02305; 4-guanidinobutanoic acid cpd: C01035; spermine cpd: C00750; 4-acetamidobutanoic acid cpd: C02946; 1-pyrroline-4-hydroxy-2-carboxylate cpd: C04282PathwayMetabolic pathway nameTotalThe number of metabolites in this pathwayHitsThe number of differential metabolites hit this pathwayRawpP value of metabolic pathway enrichment analysis−ln(p)Minus log base E of PHolm adjustP values corrected by Holm–Bonferroni method for multiple hypothesis testingFDRP value corrected by false discovery rate (FDR) method for multiple hypothesis testingImpactImpact value of metabolic pathway topology analysisHits cpdThe names and KEGG IDs of differential metabolites hit the pathwayTotal cpdAll metabolite names and KEGG IDs contained in this pathwayTable 4
Enrichment analysis results based on mass spectrometry combined with gas phase.
PathwayTotalHitsRawp−ln(p)Holm adjustFDRImpactHits cpdTotal cpdPyrimidine metabolism4150.00136.62930.110.1080.09731Deoxyuridine cpd: C00526; uracil cpd: C00106; beta-alanine cpd: C00099; thymine cpd: C00178; 3-aminoisobutanoic acid cpd: C05145Thioredoxin cpd: C00342; uridine 5′-diphosphate cpd: C00015; L-Glutamine cpd: C00064; carbamoylphosphate cpd: C00169; 4,5-dihydroorotic acid cpd: C00337; orotidylic acid cpd: C01103; RNA cpd: C00046; uridine triphosphate cpd: C00075; uridine 5′-monophosphate cpd: C00105; uridine cpd: C00299; dihydrouracil cpd: C00429; ureidopropionic acid cpd: C02642; cytidine triphosphate cpd: C00063; CDP cpd: C00112; cytidine monophosphate cpd: C00055; cytidine cpd: C00475; thioredoxin disulfide cpd: C00343; dCDP cpd: C00705; dCTP cpd: C00458; dCMP cpd: C00239; deoxycytidine cpd: C00881; deoxyuridine triphosphate cpd: C00460; dUDP cpd: C01346; dUMP cpd: C00365; deoxyuridine cpd: C00526; thymidine 5′-triphosphate cpd: C00459; dTDP cpd: C00363; 5-thymidylic acid cpd: C00364; thymidine cpd: C00214; dihydrothymine cpd: C00906; ureidoisobutyric acid cpd: C05100; P1,P4-bis(5′-uridyl) tetraphosphate cpd: C06198; ureidosuccinic acid cpd: C00438; phosphoribosyl pyrophosphate cpd: C00119; orotic acid cpd: C00295; uracil cpd: C00106; beta-alanine cpd: C00099; DNA cpd: C00039; deoxyribose 1-phosphate cpd: C00672; thymine cpd: C00178; 3-aminoisobutanoic acid cpd: C05145Pantothenate and CoA biosynthesis1520.03843.2608110Beta-alanine cpd: C00099; uracil cpd: C00106Dephospho-CoA cpd: C00882; pantetheine 4′-phosphate cpd: C01134; pantetheine cpd: C00831; 4-phosphopantothenoylcysteine cpd: C04352; D-pantothenoyl-L-cysteine cpd: C04079; d-4′-phosphopantothenate cpd: C03492; L-cysteine cpd: C00097; pantothenic acid cpd: C00864; ureidopropionic acid cpd: C02642; dihydrouracil cpd: C00429; L-valine cpd: C00183; coenzyme a cpd: C00010; beta-alanine cpd: C00099; uracil cpd: C00106; alpha-ketoisovaleric acid cpd: C00141Beta-alanine metabolism1720.04843.0283110.44444Beta-alanine cpd: C00099; uracil cpd: C00106Acrylyl-CoA cpd: C00894; 3-hydroxypropionyl-CoA cpd: C05668; hydroxypropionic acid cpd: C01013; malonyl-CoA cpd: C00083; beta-alanine cpd: C00099; L-aspartic acid cpd: C00049; spermine cpd: C00750; 1,3-diaminopropane cpd: C00986; 3-aminopropionaldehyde cpd: C05665; ureidopropionic acid cpd: C02642; dihydrouracil cpd: C00429; anserine cpd: C01262; propionyl-CoA cpd: C00100; Acetyl-CoA cpd: C00024; malonic semialdehyde cpd: C00222; spermidine cpd: C00315; uracil cpd: C00106Starch and sucrose metabolism1920.05932.8253110.07451D-maltose cpd: C00208; isomaltose cpd: C00252beta-D-fructose cpd: C02336; sucrose cpd: C00089; uridine diphosphate glucuronic acid cpd: C00167; uridine diphosphate glucose cpd: C00029; glucose 1-phosphate cpd: C00103; 3-methoxy-4-hydroxyphenylglycol glucuronide cpd: C03033; glucose 6-phosphate cpd: C00668; alpha-D-glucose cpd: C00267; starch cpd: C00369; amylose cpd: C00718; trehalose cpd: C01083; D-maltose cpd: C00208; D-glucose cpd: C00031; dextrin cpd: C00721; isomaltose cpd: C00252; beta-D-fructose 6-phosphate cpd: C05345; UDP-D-xylose cpd: C00190; alpha-D-glucose 1,6-bisphosphate cpd: C01231; D-glucuronic acid cpd: C00191Propanoate metabolism2020.0652.733110Beta-alanine cpd: C00099; 2-hydroxybutyric acid cpd: C05984(S)-methylmalonic acid semialdehyde cpd: C06002; methylmalonyl-CoA cpd: C00683; propionyl-CoA cpd: C00100; propinol adenylate cpd: C05983; R-methylmalonyl-CoA cpd: C01213; succinic acid cpd: C00042; hydroxypropionic acid cpd: C01013; beta-alanine cpd: C00099; 2-propyn-1-al cpd: C05985; acetyl-CoA cpd: C00024; malonyl-CoA cpd: C00083; 2-hydroxybutyric acid cpd: C05984; acrylyl-CoA cpd: C00894; propionic acid cpd: C00163; succinyl-CoA cpd: C00091; 3-hydroxypropionyl-CoA cpd: C05668; malonic semialdehyde cpd: C00222; propynoic acid cpd: C00804; acetoacetyl-CoA cpd: C00332; 2-ketobutyric acid cpd: C00109D-glutamine and D-glutamate metabolism510.10162.2866110Oxoglutaric acid cpd: C00026L-glutamic acid cpd: C00025; D-glutamine cpd: C00819; L-glutamine cpd: C00064; oxoglutaric acid cpd: C00026; D-glutamic acid cpd: C00217Sulfur metabolism510.10162.2866110.3Sulfate cpd: C00059Phosphoadenosine phosphosulfate cpd: C00053; adenosine phosphosulfate cpd: C00224; sulfite cpd: C00094; sulfate cpd: C00059; adenosine 3′,5′-diphosphate cpd: C00054Cyanoamino acid metabolism610.12072.1145110Glycine cpd: C000373-Cyano-L-alanine cpd: C02512; beta-aminopropionitrile cpd: C05670; glycine cpd: C00037; gamma-glutamyl-beta-cyanoalanine cpd: C05711; gamma-glutamyl-beta-aminopropiononitrile cpd: C06114; L-serine cpd: C00065Methane metabolism910.17561.7394110Glycine cpd: C00037S-formylglutathione cpd: C01031; glycine cpd: C00037; 5,10-methylene-THF cpd: C00143; S-(Hydroxymethyl)glutathione cpd: C14180; methanol cpd: C00132; formic acid cpd: C00058; L-serine cpd: C00065; formaldehyde cpd: C00067; 5-methyltetrahydrofolic acid cpd: C00440Nitrogen metabolism910.17561.7394110Glycine cpd: C00037Ammonia cpd: C00014; carbon dioxide cpd: C00011; L-glutamic acid cpd: C00025; L-glutamine cpd: C00064; L-cystathionine cpd: C02291; L-histidine cpd: C00135; carbamoylphosphate cpd: C00169; carbonic acid cpd: C01353; glycine cpd: C00037Arginine and proline metabolism4420.23821.4345110.04414Hydroxyproline cpd: C01157; 4-acetamidobutanoic acid cpd: C02946L-glutamine cpd: C00064; ammonia cpd: C00014; carbamoylphosphate cpd: C00169; ornithine cpd: C00077; citrulline cpd: C00327; L-aspartic acid cpd: C00049; argininosuccinic acid cpd: C03406; L-arginine cpd: C00062; L-glutamic acid cpd: C00025; N-acetylornithine cpd: C00437; L-proline cpd: C00148; peptide cpd: C00012; D-proline cpd: C00763; hydroxyproline cpd: C01157; pyrroline hydroxycarboxylic acid cpd: C04281; L-4-hydroxyglutamate semialdehyde cpd: C05938; L-erythro-4-hydroxyglutamate cpd: C05947; N-(o)-hydroxyarginine cpd: C05933; guanidinoacetic acid cpd: C00581; creatine cpd: C00300; gamma-aminobutyric acid cpd: C00334; agmatine cpd: C00179; L-glutamic-gamma-semialdehyde cpd: C01165; L-glutamic acid 5-phosphate cpd: C03287; (S)-1-pyrroline-5-carboxylate cpd: C03912; putrescine cpd: C00134; 4-aminobutyraldehyde cpd: C00555; S-adenosylmethioninamine cpd: C01137; S-adenosylmethionine cpd: C00019; spermidine cpd: C00315; N-Acetylputrescine cpd: C02714; N4-Acetylaminobutanal cpd: C05936; cis-4-hydroxy-D-proline cpd: C03440; Fumaric acid cpd: C00122; urea cpd: C00086; N-Acetyl-L-alanine cpd: C00624; 1-pyrroline-2-carboxylic acid cpd: C03564; D-4-hydroxy-2-oxoglutarate cpd: C05946; nitric oxide cpd: C00533; phosphocreatine cpd: C02305; 4-guanidinobutanoic acid cpd: C01035; spermine cpd: C00750; 4-acetamidobutanoic acid cpd: C02946; 1-pyrroline-4-hydroxy-2-carboxylate cpd: C04282Glycerolipid metabolism1810.32131.1355110.28098Glycerol cpd: C00116Triacylglycerol cpd: C00422; acyl-CoA cpd: C00040; 1,2-diacyl-sn-glycerol cpd: C00641; PA(16 :  : 0/16 :  : 0) cpd: C00416; 1-acyl-sn-glycerol 3-phosphate cpd: C00681; 1-acylglycerol cpd: C01885; glycerol 3-phosphate cpd: C00093; dihydroxyacetone cpd: C00184; glycerol cpd: C00116; lactaldehyde cpd: C05999; D-glyceraldehyde cpd: C00577; glyceric acid cpd: C00258; digalactosyl-diacylglycerol cpd: C06037; fatty acid cpd: C00162; dihydroxyacetone phosphate cpd: C00111; propylene glycol cpd: C00583; 3-phospho-D-glycerate cpd: C00197; 1,2-diacyl-3-beta-D-galactosyl-sn-glycerol cpd: C03692Citrate cycle (TCA cycle)2010.35011.0496110.06799Oxoglutaric acid cpd: C00026Enzyme N6-(dihydrolipoyl)lysine cpd: C15973; oxoglutaric acid cpd: C00026; thiamine pyrophosphate cpd: C00068; Enzyme N6-(lipoyl)lysine cpd: C15972; 3-carboxy-1-hydroxypropylthiamine diphosphate cpd: C05381; Succinyl-CoA cpd: C00091; succinic acid cpd: C00042; oxalosuccinic acid cpd: C05379; isocitric acid cpd: C00311; oxalacetic acid cpd: C00036; Acetyl-CoA cpd: C00024; L-malic acid cpd: C00149; cis-aconitic acid cpd: C00417; citric acid cpd: C00158; pyruvic acid cpd: C00022; 2-(a-hydroxyethyl)thiamine diphosphate cpd: C05125; [dihydrolipoyllysine-residue succinyltransferase] S-succinyldihydrolipoyllysine cpd: C16254; Fumaric acid cpd: C00122; S-acetyldihydrolipoamide-E cpd: C16255; phosphoenolpyruvic acid cpd: C00074Butanoate metabolism2210.37770.97365110Oxoglutaric acid cpd: C000263-Butyn-1-al cpd: C06145; (R)-3-hydroxybutyric acid cpd: C01089; acetoacetic acid cpd: C00164; 3-hydroxy-3-methylglutaryl-CoA cpd: C00356; Acetyl-CoA cpd: C00024; Acetoacetyl-CoA cpd: C00332; (S)-3-hydroxybutanoyl-CoA cpd: C01144; Crotonoyl-CoA cpd: C00877; gamma-aminobutyric acid cpd: C00334; L-glutamic acid cpd: C00025; Butanoyl-CoA cpd: C00136; butanal cpd: C01412; succinic acid semialdehyde cpd: C00232; butyric acid cpd: C00246; oxoglutaric acid cpd: C00026; thiamine pyrophosphate cpd: C00068; pyruvic acid cpd: C00022; 3-butynoate cpd: C06144; 1-butanol cpd: C06142; succinic acid cpd: C00042; 2-hydroxyglutarate cpd: C02630; 2-(a-hydroxyethyl)thiamine diphosphate cpd: C05125Alanine, aspartate, and glutamate metabolism2410.40420.90587110.06329Oxoglutaric acid cpd: C00026N-Acetyl-L-aspartic acid cpd: C01042; 2-oxosuccinamate cpd: C02362; L-aspartic acid cpd: C00049; D-aspartic acid cpd: C00402; argininosuccinic acid cpd: C03406; adenylsuccinic acid cpd: C03794; L-Alanine cpd: C00041; succinic acid semialdehyde cpd: C00232; L-glutamic acid cpd: C00025; gamma-aminobutyric acid cpd: C00334; L-Glutamine cpd: C00064; ammonia cpd: C00014; 2-keto-glutaramic acid cpd: C00940; (S)-1-pyrroline-5-carboxylate cpd: C03912; oxalacetic acid cpd: C00036; L-Asparagine cpd: C00152; Fumaric acid cpd: C00122; pyruvic acid cpd: C00022; ureidosuccinic acid cpd: C00438; succinic acid cpd: C00042; oxoglutaric acid cpd: C00026; carbamoylphosphate cpd: C00169; glucosamine 6-phosphate cpd: C00352; 5-phosphoribosylamine cpd: C03090Galactose metabolism2610.42960.84494110Glycerol cpd: C00116Stachyose cpd: C01613; D-tagatose 6-phosphate cpd: C01097; D-gal alpha 1->6d-gal alpha 1->6d-glucose cpd: C05404; sucrose cpd: C00089; raffinose cpd: C00492; melibiose cpd: C05402; D-galactose cpd: C00124; galactosylglycerol cpd: C05401; epimelibiose cpd: C05400; melibiitol cpd: C05399; galactinol cpd: C01235; Alpha-D-Glucose cpd: C00267; alpha-lactose cpd: C00243; glucose 1-phosphate cpd: C00103; uridine diphosphategalactose cpd: C00052; uridine diphosphate glucose cpd: C00029; galactose 1-phosphate cpd: C00446; glucose 6-phosphate cpd: C00668; D-tagatose 1,6-bisphosphate cpd: C03785; D-Fructose cpd: C00095; D-glucose cpd: C00031; galactitol cpd: C01697; glycerol cpd: C00116; D-Mannose cpd: C00159; sorbitol cpd: C00794; myoinositol cpd: C00137Glutathione metabolism2610.42960.84494110.00573Glycine cpd: C00037R-S-cysteinylglycine cpd: C05729; R-S-glutathione cpd: C02320; glutathione cpd: C00051; NADP cpd: C00006; NADPH cpd: C00005; oxidized glutathione cpd: C00127; gamma-glutamylcysteine cpd: C00669; glycine cpd: C00037; L-cysteine cpd: C00097; L-glutamic acid cpd: C00025; cysteinylglycine cpd: C01419; pyroglutamic acid cpd: C01879; L-amino acid cpd: C00151; 5-L-glutamyl-L-alanine cpd: C03740; RX cpd: C01322; ornithine cpd: C00077; putrescine cpd: C00134; spermidine cpd: C00315; cadaverine cpd: C01672; tryparedoxin cpd: C16663; trypanothione cpd: C02090; S-substituted L-cysteine cpd: C05726; spermine cpd: C00750; aminopropylcadaverine cpd: C16565; tryparedoxin disulfide cpd: C16664; trypanothione disulfide cpd: C03170Fatty acid elongation in mitochondria2710.44190.8167110Palmitic acid cpd: C00249Palmityl-CoA cpd: C00154; (2e)-hexadecenoyl-CoA cpd: C05272; (S)-3-hydroxyhexadecanoyl-CoA cpd: C05258; 3-oxohexadecanoyl-CoA cpd: C05259; Acetyl-CoA cpd: C00024; Tetradecanoyl-CoA cpd: C02593; (2e)-tetradecenoyl-CoA cpd: C05273; (S)-3-hydroxytetradecanoyl-CoA cpd: C05260; 3-oxotetradecanoyl-CoA cpd: C05261; Lauroyl-CoA cpd: C01832; (2e)-dodecenoyl-CoA cpd: C03221; (S)-3-hydroxydodecanoyl-CoA cpd: C05262; 3-oxododecanoyl-CoA cpd: C05263; Decanoyl-CoA (n-C10: 0CoA) cpd: C05274; (2e)-decenoyl-CoA cpd: C05275; (S)-hydroxydecanoyl-CoA cpd: C05264; 3-oxodecanoyl-CoA cpd: C05265; Octanoyl-CoA cpd: C01944; (2e)-octenoyl-CoA cpd: C05276; (S)-hydroxyoctanoyl-CoA cpd: C05266; 3-oxooctanoyl-CoA cpd: C05267; Hexanoyl-CoA cpd: C05270; trans-2-hexenoyl-CoA cpd: C05271; (S)-hydroxyhexanoyl-CoA cpd: C05268; 3-oxohexanoyl-CoA cpd: C05269; Butanoyl-CoA cpd: C00136; palmitic acid cpd: C00249Porphyrin and chlorophyll metabolism2710.44190.8167110Glycine cpd: C00037Cobinamide cpd: C05774; heme O cpd: C15672; heme cpd: C00032; glycine cpd: C00037; bilirubin diglucuronide cpd: C05787; Cob(I)yrinate a,c diamide cpd: C06505; Fe2 + cpd: C14818; hemoglobin cpd: C01708; biliverdin cpd: C00500; protoporphyrinogen IX cpd: C01079; coproporphyrin III cpd: C03263; uroporphyrinogen III cpd: C01051; hydroxymethylbilane cpd: C01024; porphobilinogen cpd: C00931; 5-aminolevulinic acid cpd: C00430; uroporphyrinogen I cpd: C05766; L-Glutamic acid cpd: C00025; bilirubin cpd: C00486; protoporphyrin IX cpd: C02191; adenosyl cobinamide cpd: C06508; heme a cpd: C15670; D-Urobilinogen cpd: C05791; adenosyl cobyrinic acid a,c diamide cpd: C06506; Fe3+ cpd: C14819; coproporphyrinogen I cpd: C05768; l-glutamyl-tRNA(Glu) cpd: C02987; cytochrome c cpd: C00524Glycine, serine, and threonine metabolism3110.48860.71625110.26884Glycine cpd: C00037L-Serine cpd: C00065; choline cpd: C00114; glyceric acid cpd: C00258; betaine cpd: C00719; guanidinoacetic acid cpd: C00581; 3-phospho-D-glycerate cpd: C00197; dimethylglycine cpd: C01026; L-cystathionine cpd: C02291; glycine cpd: C00037; phosphoserine cpd: C01005; sarcosine cpd: C00213; 5,10-methylene-THF cpd: C00143; L-threonine cpd: C00188; lipoylprotein cpd: C02051; aminoacetone cpd: C01888; tetrahydrofolic acid cpd: C00101; S-aminomethyldihydrolipoylprotein cpd: C01242; dihydrolipoylprotein cpd: C02972; D-serine cpd: C00740; betaine aldehyde cpd: C00576; hydroxypyruvic acid cpd: C00168; creatine cpd: C00300; phosphohydroxypyruvic acid cpd: C03232; L-cysteine cpd: C00097; glyoxylic acid cpd: C00048; L-2-amino-3-oxobutanoic acid cpd: C03508; pyruvic acid cpd: C00022; carbon dioxide cpd: C00011; 5-aminolevulinic acid cpd: C00430; pyruvaldehyde cpd: C00546; ammonia cpd: C00014Valine, leucine, and isoleucine degradation3810.56140.57739110Methylmalonic acid cpd: C02170Enzyme N6-(lipoyl)lysine cpd: C15972; 2-methyl-1-hydroxybutyl-ThPP cpd: C15978; enzyme N6-(dihydrolipoyl)lysine cpd: C15973; 2-methyl-1-hydroxypropyl-ThPP cpd: C15976; 3-methyl-1-hydroxybutyl-ThPP cpd: C15974; acetyl-CoA cpd: C00024; acetoacetyl-CoA cpd: C00332; acetoacetic acid cpd: C00164; 3-hydroxy-3-methylglutaryl-CoA cpd: C00356; 3-methylcrotonyl-CoA cpd: C03069; 3-hydroxyisovaleryl-CoA cpd: C05998; isovaleryl-CoA cpd: C02939; 3-methyl-2-oxovaleric acid cpd: C00671; thiamine pyrophosphate cpd: C00068; L-valine cpd: C00183; 2-methylacetoacetyl-CoA cpd: C03344; (S)-3-hydroxyisobutyrate cpd: C06001; tiglyl-CoA cpd: C03345; butyryl-CoA cpd: C00630; S-(2-methylbutanoyl)-dihydrolipoamide cpd: C15979; alpha-ketoisovaleric acid cpd: C00141; L-isoleucine cpd: C00407; R-methylmalonyl-CoA cpd: C01213; methylmalonyl-CoA cpd: C00683; propionyl-CoA cpd: C00100; (S)-methylmalonic acid semialdehyde cpd: C06002; (S)-b-aminoisobutyric acid cpd: C03284; 2-methyl-3-hydroxybutyryl-CoA cpd: C04405; (S)-3-hydroxyisobutyryl-CoA cpd: C06000; methacrylyl-CoA cpd: C03460; (S)-2-methylbutanoyl-CoA cpd: C15980; S-(2-methylpropionyl)-dihydrolipoamide-E cpd: C15977; 4-methyl-2-oxopentanoate cpd: C00233; S-(3-methylbutanoyl)-dihydrolipoamide-E cpd: C15975; L-leucine cpd: C00123; 3-methylglutaconyl-CoA cpd: C03231; succinyl-CoA cpd: C00091; methylmalonic acid cpd: C02170Fatty acid metabolism3910.57090.56053110Palmitic acid cpd: C00249Palmityl-CoA cpd: C00154; hexanoyl-CoA cpd: C05270; butanoyl-CoA cpd: C00136; acetyl-CoA cpd: C00024; (S)-3-hydroxybutanoyl-CoA cpd: C01144; cis,cis-3,6-dodecadienoyl-CoA cpd: C05280; (S)-hydroxyhexanoyl-CoA cpd: C05268; (S)-hydroxyoctanoyl-CoA cpd: C05266; octanoyl-CoA cpd: C01944; (S)-hydroxydecanoyl-CoA cpd: C05264; decanoyl-CoA (n-C10: 0CoA) cpd: C05274; (S)-3-hydroxydodecanoyl-CoA cpd: C05262; lauroyl-CoA cpd: C01832; primary alcohol cpd: C00226; glutaryl-CoA cpd: C00527; (S)-3-hydroxytetradecanoyl-CoA cpd: C05260; tetradecanoyl-CoA cpd: C02593; fatty acid cpd: C00162; (S)-3-hydroxyhexadecanoyl-CoA cpd: C05258; palmitic acid cpd: C00249; (2E)-hexadecenoyl-CoA cpd: C05272; trans-2-hexenoyl-CoA cpd: C05271; crotonoyl-CoA cpd: C00877; acetoacetyl-CoA cpd: C00332; trans,cis-Lauro-2,6-dienoyl-CoA cpd: C05279; coenzyme A cpd: C00010; 3-oxohexanoyl-CoA cpd: C05269; 3-oxooctanoyl-CoA cpd: C05267; (2E)-octenoyl-CoA cpd: C05276; 3-oxodecanoyl-CoA cpd: C05265; (2E)-decenoyl-CoA cpd: C05275; 3-oxododecanoyl-CoA cpd: C05263; (2E)-dodecenoyl-CoA cpd: C03221; aldehyde cpd: C00071; 3-oxotetradecanoyl-CoA cpd: C05261; (2E)-tetradecenoyl-CoA cpd: C05273; omega-hydroxy fatty acid cpd: C03547; 3-oxohexadecanoyl-CoA cpd: C05259; L-palmitoylcarnitine cpd: C02990Tryptophan metabolism4010.58030.5443110N-acetylserotonin cpd: C00978L-tryptophan cpd: C00078; melatonin cpd: C01598; serotonin cpd: C00780; 5-hydroxykynurenamine cpd: C05638; 5-hydroxykynurenine cpd: C05651; 5-hydroxy-L-tryptophan cpd: C00643; L-formylkynurenine cpd: C02700; acetoacetyl-CoA cpd: C00332; (S)-3-hydroxybutanoyl-CoA cpd: C01144; crotonoyl-CoA cpd: C00877; glutaryl-CoA cpd: C00527; oxoadipic acid cpd: C00322; 2-amino-3-carboxymuconic acid semialdehyde cpd: C04409; 3-hydroxyanthranilic acid cpd: C00632; L-kynurenine cpd: C00328; formylanthranilic acid cpd: C05653; L-3-hydroxykynurenine cpd: C03227; 3-hydroxykynurenamine cpd: C05636; indoleacetaldehyde cpd: C00637; 5-hydroxy-N-formylkynurenine cpd: C05648; 5-hydroxyindoleacetaldehyde cpd: C05634; tryptamine cpd: C00398; indoleacrylic acid cpd: C00331; acetyl-N-formyl-5-methoxykynurenamine cpd: C05642; 6-hydroxymelatonin cpd: C05643; N-acetylserotonin cpd: C00978; formyl-5-hydroxykynurenamine cpd: C05647; 4,6-dihydroxyquinoline cpd: C05639; acetyl-CoA cpd: C00024; 2-aminomuconic acid semialdehyde cpd: C03824; 2-aminobenzoic acid cpd: C00108; L-tryptophanyl-tRNA(Trp) cpd: C03512; cinnavalininate cpd: C05640; 4-(2-amino-3-hydroxyphenyl)-2,4-dioxobutanoic acid cpd: C05645; 4-(2-aminophenyl)-2,4-dioxobutanoic acid cpd: C01252; 4,8-dihydroxyquinoline cpd: C05637; indoleacetic acid cpd: C00954; 5-hydroxyindoleacetic acid cpd: C05635; N-methylserotonin cpd: C06212; N-methyltryptamine cpd: C06213Biosynthesis of unsaturated fatty acids4210.59830.51359110Palmitic acid cpd: C00249(13Z,16Z)-docosadi-13,16-enoyl-CoA cpd: C16645; tetracosenoyl-CoA cpd: C16532; docosenoyl-CoA cpd: C16531; icosenoyl-CoA cpd: C16530; tetracosanoyl-CoA cpd: C16529; docosanoyl-CoA cpd: C16528; (7Z,10Z,13Z,16Z)-docosatetraenoyl-CoA cpd: C16170; (11Z,14Z)-icosadienoyl-CoA cpd: C16180; (7Z,10Z,13Z,16Z,19Z)-docosapentaenoyl-CoA cpd: C16166; (11Z,14Z,17Z)-icosatrienoyl-CoA cpd: C16179; palmityl-CoA cpd: C00154; stearoyl-CoA cpd: C00412; eicosanoyl-CoA cpd: C02041; oleoyl-CoA cpd: C00510; linoleoyl-CoA cpd: C02050; arachidonyl-CoA cpd: C02249; 8,11,14-eicosatrienoyl-CoA cpd: C03595; gamma-linolenoyl-CoA cpd: C03035; (4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoyl-CoA cpd: C16169; (5Z,8Z,11Z,14Z,17Z)-icosapentaenoyl-CoA cpd: C16165; alpha-linolenoyl-CoA cpd: C16162; 13,16-docosadienoic acid cpd: C16533; nervonic acid cpd: C08323; erucic acid cpd: C08316; icosenoic acid cpd: C16526; tetracosanoic acid cpd: C08320; behenic acid cpd: C08281; 7,10,13,16-docosatetraenoic acid cpd: C16527; icosadienoic acid cpd: C16525; clupanodonic acid cpd: C16513; icosatrienoic acid cpd: C16522; palmitic acid cpd: C00249; stearic acid cpd: C01530; arachidic acid cpd: C06425; oleic acid cpd: C00712; linoleic acid cpd: C01595; arachidonic acid cpd: C00219; 8,11,14-eicosatrienoic acid cpd: C03242; gamma-linolenic acid cpd: C06426; (4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoic acid cpd: C06429; eicosapentaenoic acid cpd: C06428; alpha-linolenic acid cpd: C06427Fatty acid biosynthesis4310.60710.49905110Palmitic acid cpd: C00249Tetradecanoyl-[acp] cpd: C05761; malonyl-[acyl-carrier protein] cpd: C01209; hexadecanoyl-[acp] cpd: C05764; octadecanoyl-[acyl-carrier protein] cpd: C04088; oleoyl-[acyl-carrier protein] cpd: C01203; dodecanoyl-[acyl-carrier protein] cpd: C05223; decanoyl-[acp] cpd: C05755; octanoyl-[acp] cpd: C05752; Hexanoyl-[acp] cpd: C05749; butyryl-[acp] cpd: C05745; trans-hexadec-2-enoyl-[acp] cpd: C05763; (3R)-3-hydroxypalmitoyl-[acyl-carrier protein] cpd: C04633; 3-oxohexadecanoyl-[acp] cpd: C05762; trans-tetradec-2-enoyl-[acp] cpd: C05760; (3R)-3-hydroxytetradecanoyl-[acyl-carrier protein] cpd: C04688; 3-oxotetradecanoyl-[acp] cpd: C05759; trans-dodec-2-enoyl-[acp] cpd: C05758; (R)-3-hydroxydodecanoyl-[acp] cpd: C05757; 3-oxododecanoyl-[acp] cpd: C05756; trans-Dec-2-enoyl-[acp] cpd: C05754; (3R)-3-hydroxydecanoyl-[acyl-carrier protein] cpd: C04619; 3-oxodecanoyl-[acp] cpd: C05753; trans-Oct-2-enoyl-[acp] cpd: C05751; (3R)-3-hydroxyoctanoyl-[acyl-carrier protein] cpd: C04620; 3-oxooctanoyl-[acp] cpd: C05750; trans-Hex-2-enoyl-[acp] cpd: C05748; (R)-3-hydroxyhexanoyl-[acp] cpd: C05747; 3-oxohexanoyl-[acp] cpd: C05746; but-2-enoyl-[acyl-carrier protein] cpd: C04246; (3R)-3-hydroxybutanoyl-[acyl-carrier protein] cpd: C04618; acetyl-[acyl-carrier protein] cpd: C03939; Malonyl-CoA cpd: C00083; acetyl-CoA cpd: C00024; acyl-carrier protein cpd: C00229; Holo-[carboxylase] cpd: C06250; carboxybiotin-carboxyl-carrier protein cpd: C04419; myristic acid cpd: C06424; 3-oxostearoyl-[acp] cpd: C16219; stearic acid cpd: C01530; oleic acid cpd: C00712; dodecanoic acid cpd: C02679; palmitic acid cpd: C00249; acetoacetyl-[acp] cpd: C05744Primary bile acid biosynthesis4610.63230.45839110.02976Glycine cpd: C00037Cholesterol cpd: C00187; cholest-5-ene-3beta,26-diol cpd: C15610; 25-hydroxycholesterol cpd: C15519; 7 alpha,26-dihydroxy-4-cholesten-3-one cpd: C17336; 4-cholesten-7alpha,12alpha-diol-3-one cpd: C17339; 7a-hydroxy-cholestene-3-one cpd: C05455; 5-b-cholestane-3a , 7a ,12a-triol cpd: C05454; (25R)-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-26-oyl-CoA cpd: C15613; (25S)-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-26-oyl-CoA cpd: C17343; 3a,7a,12a-trihydroxy-5b-cholest-24-enoyl-CoA cpd: C05460; 3a,7a,12a-trihydroxy-5b-24-oxocholestanoyl-CoA cpd: C05467; 3a,7a-dihydroxy-5b-24-oxocholestanoyl-CoA cpd: C05449; chenodeoxycholoyl-CoA cpd: C05337; glycine cpd: C00037; taurine cpd: C00245; 3alpha,7alpha,12alpha,26-tetrahydroxy-5beta-cholestane cpd: C05446; 3a,7a,12a-trihydroxy-5b-cholestan-26-al cpd: C01301; 3 beta,7 alpha-dihydroxy-5-cholestenoate cpd: C17335; 7-a,27-dihydroxycholesterol cpd: C06341; 7a-Hydroxycholesterol cpd: C03594; 7-a,25-dihydroxycholesterol cpd: C15520; Choloyl-CoA cpd: C01794; 24-hydroxycholesterol cpd: C13550; 3alpha,7alpha-dihydroxy-5beta-cholestanate cpd: C04554; 3a,7a-dihydroxy-5b-cholestane cpd: C05452; 3 alpha,7 alpha,26-trihydroxy-5beta-cholestane cpd: C05444; 3a,7a-dihydroxy-5b-cholestan-26-al cpd: C05445; (25R)-3alpha,7alpha-dihydroxy-5beta-cholestanoyl-CoA cpd: C17345; (25S)-3alpha,7alpha-dihydroxy-5beta-cholestanoyl-CoA cpd: C17346; 3a,7a-dihydroxy-5b-cholest-24-enoyl-CoA cpd: C05447; 3a,7a,12a-Trihydroxy-5b-cholestanoic acid cpd: C04722; 3a,7a,12a-trihydroxy-5b-cholestanoyl-CoA cpd: C05448; 3a,7a,12a,24-tetrahydroxy-5b-cholestanoyl-CoA cpd: C05450; 3 beta-hydroxy-5-cholestenoate cpd: C17333; (24S)-cholest-5-ene-3beta,7alpha,24-triol cpd: C15518; 7 alpha-hydroxy-3-oxo-4-cholestenoate cpd: C17337; 7a,12a-dihydroxy-5b-cholestan-3-one cpd: C05453; 7a-hydroxy-5b-cholestan-3-one cpd: C05451; chenodeoxycholic acid cpd: C02528; chenodeoxycholic acid glycine conjugate cpd: C05466; taurochenodesoxycholic acid cpd: C05465; 7alpha,25-dihydroxy-4-cholesten-3-one cpd: C17332; cholic acid cpd: C00695; glycocholic acid cpd: C01921; taurocholic acid cpd: C05122; 7 alpha,24-dihydroxy-4-cholesten-3-one cpd: C17331Purine metabolism6810.77490.25507110Sulfate cpd: C00059Guanosine diphosphate cpd: C00035; xanthine cpd: C00385; D-ribulose 5-phosphate cpd: C00117; phosphoribosyl pyrophosphate cpd: C00119; L-glutamine cpd: C00064; 5-phosphoribosylamine cpd: C03090; glycineamideribotide cpd: C03838; phosphoribosylformylglycineamidine cpd: C04640; AICAR cpd: C04677; SAICAR cpd: C04823; 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate cpd: C04751; RNA cpd: C00046; cyclic AMP cpd: C00575; adenosine triphosphate cpd: C00002; dATP cpd: C00131; ADP cpd: C00008; dADP cpd: C00206; adenosine monophosphate cpd: C00020; adenylsuccinic acid cpd: C03794; inosinic acid cpd: C00130; adenosine cpd: C00212; deoxyadenosine monophosphate cpd: C00360; deoxyadenosine cpd: C00559; deoxyinosine cpd: C05512; xanthosine cpd: C01762; IDP cpd: C00104; guanosine monophosphate cpd: C00144; xanthylic acid cpd: C00655; hypoxanthine cpd: C00262; inosine cpd: C00294; guanine cpd: C00242; deoxyguanosine cpd: C00330; allantoic acid cpd: C00499; uric acid cpd: C00366; 5-hydroxyisourate cpd: C11821; guanosine 3′-diphosphate 5′-triphosphate cpd: C04494; guanosine triphosphate cpd: C00044; 2′-deoxyguanosine 5′-monophosphate cpd: C00362; dGDP cpd: C00361; guanosine cpd: C00387; dGTP cpd: C00286; cyclic GMP cpd: C00942; sulfate cpd: C00059; adenosine phosphosulfate cpd: C00224; phosphoadenosine phosphosulfate cpd: C00053; 5′-Phosphoribosyl-N-formylglycinamide cpd: C04376; inosine triphosphate cpd: C00081; xanthosine 5-triphosphate cpd: C00700; diadenosine tetraphosphate cpd: C01260; P1,P4-bis(5′-xanthosyl) tetraphosphate cpd: C04392; adenosine diphosphate ribose cpd: C00301; adenine cpd: C00147; dIDP cpd: C01344; 2′-deoxyinosine triphosphate cpd: C01345; diadenosine triphosphate cpd: C06197; phosphoribosyl formamidocarboxamide cpd: C04734; 5-hydroxy-2-oxo-4-ureido-2,5-dihydro-1H-imidazole-5-carboxylate cpd: C12248; 5-aminoimidazole ribonucleotide cpd: C03373; DNA cpd: C00039; ammonia cpd: C00014; urea cpd: C00086; (S)-ureidoglycolic acid cpd: C00603; guanosine 3′,5′-bis(diphosphate) cpd: C01228; adenosine 3′,5′-diphosphate cpd: C00054; diguanosine tetraphosphate cpd: C01261; dIMP cpd: C06196; 5-amino-4-imidazolecarboxyamide cpd: C04051; (S) (+)-allantoin cpd: C02350Aminoacyl-tRNA biosynthesis6910.77990.24863110Glycine cpd: C00037L-asparagine cpd: C00152; tRNA(Asn) cpd: C01637; L-histidine cpd: C00135; tRNA(His) cpd: C01643; L-phenylalanine cpd: C00079; tRNA(Phe) cpd: C01648; L-arginine cpd: C00062; tRNA(Arg) cpd: C01636; L-glutamine cpd: C00064; tRNA(Gln) cpd: C01640; L-cysteine cpd: C00097; tRNA(Cys) cpd: C01639; glycine cpd: C00037; tRNA(Gly) cpd: C01642; tRNA(Asp) cpd: C01638; L-aspartic acid cpd: C00049; L-serine cpd: C00065; tRNA(Ser) cpd: C01650; L-methionine cpd: C00073; tRNA(Met) cpd: C01647; L-valine cpd: C00183; tRNA(Val) cpd: C01653; L-alanine cpd: C00041; tRNA(Ala) cpd: C01635; L-lysine cpd: C00047; tRNA(Lys) cpd: C01646; L-isoleucine cpd: C00407; tRNA(Ile) cpd: C01644; tRNA(Leu) cpd: C01645; L-leucine cpd: C00123; L-threonine cpd: C00188; tRNA(Thr) cpd: C01651; tRNA(Trp) cpd: C01652; L-tryptophan cpd: C00078; N10-formyl-THF cpd: C00234; L-methionyl-tRNA cpd: C02430; L-tyrosine cpd: C00082; tRNA(Tyr) cpd: C00787; L-proline cpd: C00148; tRNA(Pro) cpd: C01649; L-glutamic acid cpd: C00025; tRNA(Glu) cpd: C01641; glutaminyl-tRNA cpd: C02282; L-asparaginyl-tRNA(Asn) cpd: C03402; tRNA(Sec) cpd: C16636; L-seryl-tRNA(Sec) cpd: C06481; O-phosphoseryl-tRNA(Sec) cpd: C16638; L-histidyl-tRNA(His) cpd: C02988; L-phenylalanyl-tRNA(Phe) cpd: C03511; L-arginyl-tRNA(Arg) cpd: C02163; L-cysteinyl-tRNA(Cys) cpd: C03125; Glycyl-tRNA(Gly) cpd: C02412; L-aspartyl-tRNA(Asp) cpd: C02984; L-seryl-tRNA(Ser) cpd: C02553; L-valyl-tRNA(Val) cpd: C02554; L-alanyl-tRNA cpd: C00886; L-lysyl-tRNA cpd: C01931; L-isoleucyl-tRNA(Ile) cpd: C03127; L-leucyl-tRNA cpd: C02047; L-threonyl-tRNA(Thr)cpd: C02992; L-tryptophanyl-tRNA(Trp) cpd: C03512; tetrahydrofolic acid cpd: C00101; N-formylmethionyl-tRNA cpd: C03294; L-tyrosyl-tRNA(Tyr) cpd: C02839; L-prolyl-tRNA(Pro) cpd: C02702; L-glutamyl-tRNA(Glu) cpd: C02987; L-glutamyl-tRNA(Gln) cpd: C06112; L-aspartyl-tRNA(Asn) cpd: C06113; L-selenocysteinyl-tRNA(Sec) cpd: C06482PathwayMetabolic pathway nameTotalThe number of metabolites in this pathwayHitsThe number of differential metabolites hit this pathwayRawpP value of metabolic pathway enrichment analysis−ln (p)Minus log base E of PHolm adjustP values corrected by Holm–Bonferroni method for multiple hypothesis testingFDRP value corrected by false discovery rate (FDR) method for multiple hypothesis testingImpactImpact value of metabolic pathway topology analysisHits cpdThe names and KEGG IDs of differential metabolites hit the pathwayTotal cpdAll metabolite names and KEGG IDs contained in this pathwayFigure 3
Metabolic pathway enrichment analysis of differential metabolites. (a) Pathway analysis of group M versus group T based on mass spectrometry combined with high-performance liquid chromatography. (b) Pathway analysis of group M versus group T based on mass spectrometry combined with gas chromatography. Each bubble represents a metabolic pathway; the bubble size represents the influence factor, the larger the size, the larger the influence factor; the vertical coordinate of the bubble and the color of the bubble indicate theP value of the enrichment analysis; the darker the color, the smaller the P value, the more significant the enrichment.
(a)(b)(c)


## 3.4. Comparison of Serum Inflammatory Factor Levels in Three Groups of Mice

Finally, we observed the serum inflammatory factor levels in the three groups of mice to determine the effect of Yanghe decoction on inflammatory factors in mice with Hashimoto’s thyroiditis. The results showed that the serum inflammatory factor IL-35 was significantly lower and IL-6 was significantly higher in mice of group M than group C, while the serum inflammatory factor IL-35 was significantly higher and IL-6 was significantly lower in mice of group T than group M. The results showed that the serum inflammatory factor IL-35 was significantly lower and IL-6 was significantly lower in mice of group T than group C. See Figure4.Figure 4
Comparison of serum levels of inflammatory factors IL-35 and IL-6 in three groups of mice.


## 4. Discussion

Metabolism plays a central role as a signaling molecule, immunomodulator, endogenous toxin, and environmental sensor in all areas of biology, from ecology to bioengineering to cancer. Each of these fields is now increasingly being studied from a metabolic perspective. And these studies are valuable from a big picture perspective [11]. The metabolome is a collection of small-molecule chemical entities involved in metabolism and has traditionally been studied to identify biomarkers for the diagnosis and prediction of disease. Nowadays, the value of metabolomic analysis has been redefined from a simple biomarker identification tool to a technique for discovering active drivers of biological processes [12]. Metabolomics is the high-throughput characterization of metabolites from cells, organs, tissues, or biofluids using advanced analytical chemistry techniques [13]. NMR and mass spectrometry are commonly used in metabolomics; NMR is highly reproducible and quantitative, has a simple sample preparation protocol, and is capable of measuring analytes in a variety of solvent conditions, but it has low sensitivity. In contrast, the high sensitivity and low detection limits of mass spectrometry enable the detection of subtle metabolic changes that are not visible with NMR [14]. In this experiment, a total of 38 differential metabolites were screened based on mass spectrometry coupled with the high-performance liquid phase, 120 differential metabolites were screened based on mass spectrometry coupled with gas phase, and a total of α-glucose and β-glucose were analyzed based on NMR test results.Measuring metabolite concentrations by metabolomics only tells half the story. Equally important is to understand pathway activity, which can be quantified as the flow of material per unit time, i.e., metabolic flux. In this experiment, we took intersections of pathways enriched by differential metabolites in the M and T groups for both mass spectrometry coupled with high-performance liquid and gas phases, yielding a total of five common pathways, namely, amino acid tRNA biosynthesis, D-glutamine and D-glutamate metabolism, tryptophan metabolism, nitrogen metabolism, and arginine and proline metabolism.Amino tRNA is a substrate for translation and plays a key role in determining how the genetic code is interpreted into amino acids. The function of aminyl-tRNA synthesis is to precisely match amino acids to tRNAs containing the corresponding anticodons [15]. Aminyl-tRNA synthetase is essential for the physical interpretation of the genetic code [16], and in addition to its function in protein synthesis, it is involved in various cellular processes such as immune and inflammatory responses, angiogenesis, and apoptosis [17,18]. Just one study showed that the pentose phosphate pathway, amyl-tRNA biosynthesis, and pyrimidine metabolism are the main pathways altered in hypothyroidism [19]. Glutamate is a key excitatory neurotransmitter responsible for maintaining cognitive function and neuronal plasticity [20], while metabolites associated with glutamate metabolism, 2-ketoglutarate, L-aspartate, and fumarate are associated with the gut microbiota, and their alterations may affect human health [21]. Some studies have indicated that glycerophospholipid, glutamine, and glutamate metabolism, and related metabolites are potential key targets for common molecular mechanisms linking HIV to NCDs through inflammation and oxidative stress [22]. Tryptophan is an essential aromatic amino acid consisting of a β-carbon attached to the 3-position of the indole group. Although tryptophan is the least abundant amino acid in proteins and cells, it is a biosynthetic precursor for a large number of microbial and host metabolites [23,24]. Tryptophan metabolism in the intestine is the direct conversion of tryptophan by intestinal microorganisms into several molecules, such as indoles and their derivatives. And many of these indole derivatives, in turn, are ligands for aryl hydrocarbon receptors [25]. Aryl hydrocarbon receptor signaling is thought to be a key component of the immune response at the barrier site and thus can maintain intestinal homeostasis by acting on epithelial renewal, barrier integrity, and many immune cell types [26]. Arginine is a nonessential or semiessential amino acid that plays an important role in a variety of biological functions including cell proliferation, survival, and protein synthesis. It is also a precursor for the production of nitric oxide, polyamines, proline, creatinine, and glutamate. As a multifunctional amino acid, arginine plays an important role in physical health by being involved in tissue damage and chronic metabolic diseases [27]. Arginine has also been associated with endothelial function, inflammation, and airway hyperresponsiveness [28]. Just one study indicated that arginine and proline metabolic pathways are related to asthma pathogenesis [29]. The above combined with the results of the present experiment could suggest that Yanghe decoction may maintain homeostasis by altering the relevant metabolic pathways and thus improving the disease. In addition, the above pathways have shown relevance to inflammation as well as immune cells. For this reason, we also investigated the effect of Yanghe decoction.Inflammation is a comprehensive physiological response to tissue damage, which is caused by physical injury, infection, exposure to toxins, or other types of trauma [30]. There is growing evidence that inflammation is a major factor in the progression of many diseases, including autoimmune thyroiditis [31]. The results of the present study showed that serum inflammatory factor IL-35 was significantly lower and IL-6 was significantly higher in mice in the model group compared with the normal control group, whereas serum inflammatory factor IL-35 was significantly higher and IL-6 was significantly lower in mice treated based on Yanghe decoction compared with the model group. This may be due to the effect of one or a combination of herbs in the composition of Yanghe decoction. Deer antler tablets have antifatigue, anti-inflammatory, and analgesic effects, while deer antler peptides are the main active ingredients obtained by isolation from deer antler tablets [32]. It has been shown that in osteoblasts, antler peptides block TNF-α-mediated inhibition of osteoblastogenesis and inhibit osteoclastogenesis through the nuclear factor-κB (NF-κB)/p65 pathway. In addition, deer antler peptides reduce levels of interleukin 1β and interleukin 6, as well as oxidative responses induced by increased catalase activity and reduced malondialdehyde levels [33]. Previous studies have also indicated that Yanghe decoction may improve the symptoms of Hashimoto’s thyroiditis and reduce inflammation [34]. This could suggest that Yanghe decoction can effectively reduce the inflammatory response in Hashimoto’s thyroiditis, but whether the specific mechanism is related to metabolic pathways remains to be explored.


## 5. Conclusion

In summary, we detected multiple metabolites that can be altered by Yanghe decoction by NMR and mass spectrometry coupled with gas or liquid chromatography, and most of them were related to aminyl-tRNA biosynthesis, D-glutamine and D-glutamine metabolism, tryptophan metabolism, nitrogen metabolism, and arginine and proline metabolic pathways. In addition, Yanghe decoction can effectively reduce serum inflammatory factor levels in mice with Hashimoto’s thyroiditis.Although we identified the metabolites that can be altered by Yanghe decoction and the pathways that are highly affected, there are still shortcomings in this study. We only observed the metabolomic profile of plasma, and further studies should evaluate serum, urine, cerebrospinal fluid, and brain samples to accurately reflect the pathological changes in Hashimoto’s thyroiditis and the therapeutic mechanisms of Yanghe decoction.



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*Source: 6215573-2022-07-15.xml*