UHPLC-HRMS-based Multiomics to Explore the Potential Mechanisms and Biomarkers for Colorectal Cancer.

BACKGROUND: Understanding the metabolic changes in colorectal cancer (CRC) and exploring potential diagnostic biomarkers is crucial for elucidating its pathogenesis and reducing mortality. Cancer cells are typically derived from cancer tissues and can be easily obtained and cultured. Systematic studies on CRC cells at different stages are still lacking. Additionally, there is a need to validate our previous findings from human serum. METHODS: Ultrahigh-performance liquid chromatography tandem high-resolution mass spectrometry (UHPLC-HRMS)-based metabolomics and lipidomics were employed to comprehensively measure metabolites and lipids in CRC cells at four different stages and serum samples from normal control (NR) and CRC subjects. Univariate and multivariate statistical analyses were applied to select the differential metabolites and lipids between groups. Biomarkers with good diagnostic efficacy for CRC that existed in both cells and serum were screened by the receiver operating characteristic curve (ROC) analysis. Furthermore, potential biomarkers were validated using metabolite standards. RESULTS: Metabolite and lipid profiles differed significantly among CRC cells at stages A, B, C, and D. Dysregulation of glycerophospholipid (GPL), fatty acid (FA), and amino acid (AA) metabolism played a crucial role in the CRC progression, particularly GPL metabolism dominated by phosphatidylcholine (PC). A total of 46 differential metabolites and 29 differential lipids common to the four stages of CRC cells were discovered. Eight metabolites showed the same trends in CRC cells and serum from CRC patients compared to the control groups. Among them, palmitoylcarnitine and sphingosine could serve as potential biomarkers with the values of area under the curve (AUC) more than 0.80 in the serum and cells. Their panel exhibited excellent performance in discriminating CRC cells at different stages from normal cells (AUC = 1.00). CONCLUSIONS: To our knowledge, this is the first research to attempt to validate the results of metabolism studies of serum from CRC patients using cell models. The metabolic disorders of PC, FA, and AA were closely related to the tumorigenesis of CRC, with PC being the more critical factor. The panel composed of palmitoylcarnitine and sphingosine may act as a potential biomarker for the diagnosis of CRC, aiding in its prevention.

Serum untargeted lipidomics by UHPLC-ESI-HRMS aids the biomarker discovery of colorectal adenoma.

BACKGROUND: Colorectal adenoma (CA) is an important precancerous lesion and early screening target of colorectal cancer (CRC). Lipids with numerous physiological functions are proved to be involved in the development of CRC. However, there is no lipidomic study with large-scale serum samples on diagnostic biomarkers for CA. METHODS: The serum lipidomics of CA patients (n = 50) and normal control (NR) (n = 50) was performed by ultra high performance liquid chromatography-high resolution mass spectrometry with electrospray ionization (UHPLC-ESI-HRMS). Univariate and multivariate statistical analyses were utilized to screen the differential lipids between groups, and combining the constituent ratio analysis and diagnostic efficiency evaluation by receiver operating characteristic (ROC) curve disclosed the potential mechanism and biomarkers for CA. RESULTS: There were obvious differences in serum lipid profiles between CA and NR groups. Totally, 79 differential lipids were selected by criterion of P < 0.05 and fold change > 1.5 or < 0.67. Triacylglycerols (TAGs) and phosphatidylcholines (PCs) were the major differential lipids with ratio > 60%, indicating these two lipid metabolic pathways showed evident disequilibrium, which could contribute to CA formation. Of them, 12 differential lipids had good diagnostic ability as candidate biomarkers for CA (AUC ≥ 0.900) by ROC analysis. CONCLUSIONS: To our knowledge, this is the first attempt to profile serum lipidomics and explore lipid biomarkers of CA to help early screening of CRC. 12 differential lipids are obtained to act as potential diagnostic markers of CA. PCs and fatty acids were the main dysregulated biomarkers for CA in serum.

Serum Untargeted UHPLC-HRMS-Based Lipidomics to Discover the Potential Biomarker of Colorectal Advanced Adenoma.

BACKGROUND: As a key precancerous lesion, colorectal advanced adenoma (CAA) is closely related to the occurrence and development of colorectal cancer (CRC). Effective identification of CAA-related biomarkers can prevent CRC morbidity and mortality. Lipids, as an important endogenous substance, have been proved to be involved in the occurrence and development of CRC. Lipidomics is an advanced technique that studies lipid metabolism and biomarkers of diseases. However, there are no lipidomics studies based on large serum samples to explore diagnostic biomarkers for CAA. METHODS: An integrated serum lipid profile from 50 normal (NR) and 46 CAA subjects was performed using ultra-high performance liquid chromatography tandem high-resolution mass spectrometry (UHPLC-HRMS). Lipidomic data were acquired for negative and positive ionization modes, respectively. Differential lipids were selected by univariate and multivariate statistics analyses. A receiver operator characteristic curve (ROC) analysis was conducted to evaluate the diagnostic performance of differential lipids. RESULTS: A total of 53 differential lipids were obtained by combining univariate and multivariate statistical analyses (P < 0.05 and VIP > 1). In addition, 12 differential lipids showed good diagnostic performance (AUC > 0.90) for the discrimination of NR and CAA by receiver operating characteristic curve (ROC) analysis. Of them, the performance of PC 44:5 and PC 35:6e presented the outstanding performance (AUC = 1.00, (95% CI, 1.00-1.00)). Moreover, triglyceride (TAG) had the highest proportion (37.74%) as the major dysregulated lipids in the CAA. CONCLUSION: This is the first study that profiled serum lipidomics and explored lipid biomarkers with good diagnostic ability of CAA to contribute to the early prevention of CRC. Twelve differential lipids that effectively discriminate between NR and CAA serve as the potential diagnostic markers of CAA. An obvious perturbation of TAG metabolism could be involved in the CAA formation.

Proteinase K Combining Two-Step Liquid-Liquid Extraction for Plasma Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics To Discover the Potential Mechanism of Colorectal Adenoma.

LC-MS-based untargeted metabolomics have been proven to be an extremely promising technique to discover biomarkers and explore the mechanisms underlying diseases, which, however, relies heavily on sample pretreatment for metabolite extraction. In the present study, a systematic and pragmatic evaluation of eight protocols employing conventional metabolites extraction strategies, protein precipitation (PPT), and liquid-liquid extraction (LLE), with and without proteinase K (PK) incubation, was performed simultaneously, using human plasma and a mixture of 39 endogenous metabolite standards. These protocols were as follows: (1) PPT with methanol, (2) PPT with acetonitrile, (3) PPT with 2-propanol, (4) two-step LLE of CH2Cl2-MeOH, followed by MeOH-H2O, (5) PK incubation combining two-step LLE of CH2Cl2-MeOH followed by MeOH-H2O, (6) two-step LLE of CHCl3-MeOH, followed by MeOH-H2O, (7) PK incubation combining two-step LLE of CHCl3-MeOH, followed by MeOH-H2O, (8) PK incubation combining MeOH-EtOH PPT. The results suggested that two-step LLE produced broader metabolome coverage than protein precipitation, and the addition of proteinase K enhanced the extraction performance further. Taken together, PK incubation combining two-step LLE of CHCl3-MeOH, followed by MeOH-H2O, was determined to be the most suitable extraction method, because of its broad metabolome coverage, high reproducibility, and satisfactory recovery. Next, the developed optimal sample preparation method was applied successfully to profile the plasma metabolome of colorectal adenoma and uncover its potential mechanism for significant differential changes in linoleic acid and phospholipid metabolism.