Our study selected a total of 27 differential metabolites (VIP > 1.5 and FDR < 0.05) from the urine of bladder cancer patients and cancer-free controls through non-targeted metabolomic analysis. The ROC curve analysis showed that when distinguishing bladder cancer patients from cancer-free controls, the AUC value of this group of metabolites was 0.851, the sensitivity was 70%, the specificity was 89%, and the accuracy was 80%. Compared with FDA-approved urine biomarkers, this group of significantly different urinary metabolites showed better overall sensitivity and specificity than NMP-22 (69% and 77%, respectively) and BTA Track (65% and 74%, respectively) .
The ability to grade bladder cancer staging will effectively reduce unnecessary cystoscopy and overtreatment of bladder cancer . However, in this study, there was not a statistically significant difference in metabolites between NMIBC and MIBC. Sriastava's NMR study also did not find significant changes in the concentration of metabolites between bladder cancer groups . However, subsequent studies by Issaq et al. , X. Cheng , Alberice , X. Liu , and J. Peng  have shown that urinary metabolomic markers for bladder cancer indeed are able to distinguish the tumor stages. The study by Dinges et al. suggested that metabolomics may show the potential to differentiate cancer stages when studied in larger cohorts .
This group of differential metabolites may provide new insights into dysregulated urinary metabolism in bladder cancer patients. Significant changes have occurred in ten organic acids in the urine of patients with bladder cancer, including 1-methylhistidine, L-histidine, taurine, 4-acetyl aminobutyric acid, N-undecanoyl glycine, isoleucine-phenylalanine, phenylalanine-aspartic acid, valine-serine, L-arginine, and sebacic acid. The significant increase in the taurine in the urine of bladder cancer patients is consistent with the study by Wittmann et al. , that is, the taurine in the urine of bladder cancer patients is higher than that of the healthy control group. The biological background of taurine shows it to be a known free radical scavenger that can prevent cell damage and is expected to be a marker for cancer screening . Whereas leucine-phenylalanine, phenylalanine-aspartic acid, and valine-serine are collectively referred to as dipeptides, which are incomplete breakdown products of proteolytic metabolism, most of which are only transient intermediates that enter specific amino acid degradation pathways after further protein hydrolysis. Changes in amino acids and dipeptides in urine may reflect shedding into the urine due to high levels of cell division, cell death, and protein degradation. A literature search did not find any reports of dipeptides associated with bladder cancer and other cancers. The content of 1-methylhistidine in bladder cancer is increased, while the content of L-histidine is decreased. The research of Alberice and T. Zhang showed that the histidine and its methylated derivatives in the urine of patients with bladder cancer and ovarian cancer changed significantly [5, 18]. The decrease in the level of L-arginine in the urine of bladder cancer patients may be related to the catabolic disease state of cancer, resulting in increased arginine utilization, which will lead to arginine consumption . 4-Acetyl aminobutyric acid is a product of the urea cycle and amino metabolism, while N-undecanoyl glycine is a secondary metabolite of fatty acids; and metabolomics studies searched in the literature have not shown that they are associated with tumors.
The six fatty acyl groups in the urine of patients with bladder cancer are changed, including 6-hydroxyhexanoic acid, (5R)-5-hydroxyhexanoic acid, 2-hydroxyhexanoic acid, 1,11-undecanedicarboxylic acid, linoleoyl carnitine, and butenyl carnitine. The general function of acylcarnitine is to transport acyl groups (organic acids and fatty acids) from the cytoplasm to the mitochondria so that they can be broken down to produce energy, This process is known as β-oxidation. Considering that the urine under normal physiological conditions does not contain lipids, Paskenta’s study showed increased urinary acetyl carnitine and adipic acid in patients with bladder cancer, suggesting disturbances in fatty acid transport and changes in energy metabolic processes [20, 21]. Increased urinary adenosine monophosphate content and decreased ubiquinone-2 and flavin mononucleotide (FMN) in the electron chain of cellular oxygen respiration in bladder cancer patients are also considered to be associated with disturbed energy metabolism.
Mesobilirubinogen and L-urobilin are reduced in bladder cancer patients. Their metabolism is derived from the degradation of heme to produce urobilinogen, but whether their metabolic changes are related to tumors and other wasting diseases requires further research. Nicotinamide, which is a heterocyclic aromatic compound, is relatively increased in bladder cancer, and it is related to metabolic disorders of the NAD + signaling pathway (cancer) .
In this study, compared with the controls, bladder cancer patients have lower levels of dihydrotestosterone in urine. Although there are no reports of androgens metabolism changes in the literature, the research by Miyamoto et al. pointed to suggest that androgens and their receptors play a contributory role in the development of bladder cancer . The increase in urinary choline levels in bladder cancer patients is consistent with the study results by Kouznetsova et al., in which patients with advanced bladder cancer have significant multiplicative changes in urinary choline .
The change of urinary adenosine in bladder cancer patients is consistent with Jadidi-Niaragh’s study. His research pointed out that adenosine is an important factor in the tumor microenvironment that inhibits the anti-tumor response of cancer cells and immune cells. Inhibiting the production of adenosine can significantly prevent tumor growth in vivo, and targeted inhibition of adenosine receptors may be a promising therapeutic approach in tumor treatment.
Overall, our study shows the potential of a statistically significantly different set of urinary metabolites in the early detection of bladder cancer, providing new insights into changes in urinary metabolism in patients with bladder cancer. This includes changes in lipid metabolism and protein catabolism in bladder cancer patients, as well as changes in the metabolism of adenosine monophosphate (AMP), ubiquinone-2, and flavin mononucleotide (FMN) in the urine in the energy metabolism pathway. This group of differential metabolites of taurine, nicotinamide, histidine, adenosine, dihydrotestosterone, choline, and arginine has also been reported to be metabolically altered in previous tumor-related studies. However, the potential influences of diet, environment, lifestyle, medications, and other disease conditions still cannot be ruled out. Comprehensively considering multiple confounding variables and testing the effectiveness of the proposed markers are important steps in the development of markers for clinical use, which need to be verified in larger cohort studies in the future.
The advantage of using metabolomics is that it provides a functional measurement of the physiological state of an organism. Our study found that the difference between the urinary metabolites of bladder cancer patients and the cancer-free controls is statistically significant, which may provide clinically useful biomarkers for identifying metabolic changes and provide new insights into the occurrence and development of bladder cancer. In the future, more mature and standardized urinary metabolomics tests will be able to effectively supplement existing screening tools. A positive result for altered urinary metabolism prompts more detailed tests to detect and identify specific cancers or precancerous conditions.