Aihua Zhao

Serum Metabolite Profiling of Human Colorectal Cancer Using GC−TOFMS and UPLC−QTOFMS

Colorectal carcinogenesis involves the overexpression of many immediate-early response genes associated with growth and inflammation, which significantly alters downstream protein synthesis and small-molecule metabolite production. We have performed a serum metabolic analysis to test the hypothesis that the distinct metabolite profiles of malignant tumors are reflected in biofluids. In this study, we have analyzed the serum metabolites from 64 colorectal cancer (CRC) patients and 65 healthy controls using gas chromatography time-of-flight mass spectrometry (GC-TOFMS) and Acquity ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (Acquity UPLC-QTOFMS). Orthogonal partial least-squares discriminate analysis (OPLS-DA) models generated from GC-TOFMS and UPLC-QTOFMS metabolic profile data showed robust discrimination from CRC patients and healthy controls. A total of 33 differential metabolites were identified using these two analytical platforms, five of which were detected in both instruments. These metabolites potentially reveal perturbation of glycolysis, arginine and proline metabolism, fatty acid metabolism and oleamide metabolism, associated with CRC morbidity. These results suggest that serum metabolic profiling has great potential in detecting CRC and helping to understand its underlying mechanisms.

Urinary metabonomic study on colorectal cancer.

After our serum metabonomic study of colorectal cancer (CRC) patients recently published in J. Proteome Res., we profiled urine metabolites from the same group of CRC patients (before and after surgical operation) and 63 age-matched healthy volunteers using gas chromatography-mass spectrometry (GC-MS) in conjunction with a multivariate statistics technique. A parallel metabonomic study on a 1,2-dimethylhydrazine (DMH)-treated Sprague-Dawley rat model was also performed to identify significantly altered metabolites associated with chemically induced precancerous colorectal lesion. The orthogonal partial least-squares-discriminant analysis (OPLS-DA) models of metabonomic results demonstrated good separations between CRC patients or DMH-induced model rats and their healthy counterparts. The significantly increased tryptophan metabolism, and disturbed tricarboxylic acid (TCA) cycle and the gut microflora metabolism were observed in both the CRC patients and the rat model. The urinary metabolite profile of postoperative CRC subjects altered significantly from that of the preoperative stage. The significantly down-regulated gut microflora metabolism and TCA cycle were observed in postoperative CRC subjects, presumably due to the colon flush involved in the surgical procedure and weakened physical conditions of the patients. The expression of 5-hydroxytryptophan significantly decreased in postsurgery samples, suggesting a recovered tryptophan metabolism toward healthy state. Abnormal histamine metabolism and glutamate metabolism were found only in the urine samples of CRC patients, and the abnormal polyamine metabolism was found only in the rat urine. This study assessed the important metabonomic variations in urine associated with CRC and, therefore, provided baseline information complementary to serum/plasma and tissue metabonomics for the complete elucidation of the underlying metabolic mechanisms of CRC.

Ultra-performance LC/TOF MS analysis of medicinal Panax herbs for metabolomic research

In this study, metabolite profiling of five medicinal Panax herbs including Panax ginseng (Chinese ginseng), Panax notoginseng (Sanchi), Panax japonicus (Rhizoma Panacis Majoris), Panax quinquefolium L. (American ginseng), and P. ginseng (Korean ginseng) were performed using ultra-performance LC-quadrupole TOF MS (UPLC-QTOFMS) and multivariate statistical analysis technique. Principal component analysis (PCA) of the analytical data showed that the five Panax herbs could be separated into five different groups of phytochemicals. The chemical markers such as ginsenoside Rf, 20(S)-pseudoginsenoside F11, malonyl gisenoside Rb1, and gisenoside Rb2 accountable for such variations were identified through the loadings plot of PCA, and were identified tentatively by the accurate mass of TOFMS and partially verified by the available reference standards. Results from this study indicate that the proposed method is reliable for the rapid analysis of a group of metabolites present in herbal medicines and other natural products and applicable in the differentiation of complex samples that share similar chemical ingredients.

Metabolic profiling reveals disorder of amino acid metabolism in four brain regions from a rat model of chronic unpredictable mild stress

Chronic stress is closely linked to clinical depression, which could be assessed by a chronic unpredictable mild stress (CUMS) animal model. We present here a GC/MS‐based metabolic profiling approach to investigate neurochemical changes in the cerebral cortex, hippocampus, thalamus, and remaining brain tissues. Multi‐criteria assessment for multivariate statistics could identify differential metabolites between the CUMS‐model rats versus the healthy controls. This study demonstrates that the significantly perturbed metabolites mainly involving amino acids play an indispensable role in regulating neural activity in the brain. Therefore, results obtained from such metabolic profiling strategy potentially provide a unique perspective on molecular mechanisms of chronic stress.

Potential Metabolite Markers of Schizophrenia

Schizophrenia is a severe mental disorder that affects 0.5–1% of the population worldwide. Current diagnostic methods are based on psychiatric interviews, which are subjective in nature. The lack of disease biomarkers to support objective laboratory tests has been a long-standing bottleneck in the clinical diagnosis and evaluation of schizophrenia. Here we report a global metabolic profiling study involving 112 schizophrenic patients and 110 healthy subjects, who were divided into a training set and a test set, designed to identify metabolite markers. A panel of serum markers consisting of glycerate, eicosenoic acid, β-hydroxybutyrate, pyruvate and cystine was identified as an effective diagnostic tool, achieving an area under the receiver operating characteristic curve (AUC) of 0.945 in the training samples (62 patients and 62 controls) and 0.895 in the test samples (50 patients and 48 controls). Furthermore, a composite panel by the addition of urine β-hydroxybutyrate to the serum panel achieved a more satisfactory accuracy, which reached an AUC of 1 in both the training set and the test set. Multiple fatty acids and ketone bodies were found significantly (P<0.01) elevated in both the serum and urine of patients, suggesting an upregulated fatty acid catabolism, presumably resulting from an insufficiency of glucose supply in the brains of schizophrenia patients.

Salivary metabolite signatures of oral cancer and leukoplakia

Oral cancer, one of the six most common human cancers with an overall 5‐year survival rate of <50%, is often not diagnosed until it has reached an advanced stage. The aim of the current study is to explore salivary metabolomics as a disease diagnostic and stratification tool for oral cancer and leukoplakia and evaluate the potential of salivary metabolome for detection of oral squamous cell carcinoma (OSCC). Saliva metabolite profiling for a group of 37 OSCC patients, 32 oral leukoplakia (OLK) patients and 34 healthy subjects was performed using ultraperformance liquid chromatography coupled with quadrupole/time‐of‐flight mass spectrometry in conjunction with multivariate statistical analysis. The OSCC, OLK and healthy control groups demonstrate characteristic salivary metabolic signatures. A panel of five salivary metabolites including γ‐aminobutyric acid, phenylalanine, valine, n‐eicosanoic acid and lactic acid were selected using OPLS‐DA model with S‐plot. The predictive power of each of the five salivary metabolites was evaluated by receiver operating characteristic curves for OSCC. Valine, lactic acid and phenylalanine in combination yielded satisfactory accuracy (0.89, 0.97), sensitivity (86.5% and 94.6%), specificity (82.4% and 84.4%) and positive predictive value (81.6% and 87.5%) in distinguishing OSCC from the controls or OLK, respectively. The utility of salivary metabolome diagnostics for oral cancer is successfully demonstrated in this study and these results suggest that metabolomics approach complements the clinical detection of OSCC and stratifies the two types of lesions, leading to an improved disease diagnosis and prognosis.

Distinct urinary metabolic profile of human colorectal cancer.

A full spectrum of metabolic aberrations that are directly linked to colorectal cancer (CRC) at early curable stages is critical for developing and deploying molecular diagnostic and therapeutic approaches that will significantly improve patient survival. We have recently reported a urinary metabonomic profiling study on CRC subjects (n = 60) and health controls (n = 63), in which a panel of urinary metabolite markers was identified. Here, we report a second urinary metabonomic study on a larger cohort of CRC (n = 101) and healthy subjects (n = 103), using gas chromatography time-of-flight mass spectrometry and ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry. Consistent with our previous findings, we observed a number of dysregulated metabolic pathways, such as glycolysis, TCA cycle, urea cycle, pyrimidine metabolism, tryptophan metabolism, polyamine metabolism, as well as gut microbial-host co-metabolism in CRC subjects. Our findings confirm distinct urinary metabolic footprints of CRC patients characterized by altered levels of metabolites derived from gut microbial-host co-metabolism. A panel of metabolite markers composed of citrate, hippurate, p-cresol, 2-aminobutyrate, myristate, putrescine, and kynurenate was selected, which was able to discriminate CRC subjects from their healthy counterparts. A receiver operating characteristic curve (ROC) analysis of these markers resulted in an area under the receiver operating characteristic curve (AUC) of 0.993 and 0.998 for the training set and the testing set, respectively. These potential metabolite markers provide a novel and promising molecular diagnostic approach for the early detection of CRC.

Characterization of Pu-erh Tea Using Chemical and Metabolic Profiling Approaches

In this study, the chemical constituents of pu-erh tea, black tea, and green tea, as well as those of pu-erh tea products of different ages, were analyzed and compared using a chemical profiling approach. Differences in tea processing resulted in differences in the chemical constituents and the color of tea infusions. Human biological responses to pu-erh tea ingestion were also studied by using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS) in conjunction with multivariate statistical techniques. Metabolic alterations during and after pu-erh tea ingestion were characterized by increased urinary excretion of 5-hydroxytryptophan, inositol, and 4-methoxyphenylacetic acid, along with reduced excretion of 3-chlorotyrosine and creatinine. This study highlights the potential for metabonomic technology to assess nutritional interventions and is an important step toward a full understanding of pu-erh tea and its influence on human metabolism.

Metabolite Profiling of Panax notoginseng Using UPLC–ESI-MS

The metabolite profiling of different parts of Panax notoginseng was carried out using rapid ultra-performance liquid chromatography-electrospray ionization mass spectrometry (UPLC-ESI-MS) and multivariate statistical analysis. Principal component analysis (PCA) of the UPLC-ESI-MS data showed a clear separation of compositions among the flower buds, roots and rhizomes of P. notoginseng. The saponins accounting for such variations were identified through the corresponding loadings weights and were further verified by accurate mass, tandem mass and retention times of available standard saponins using UPLC quadrupole time-of-flight mass spectrometer (UPLC-QtofMS). Finally, the influential factors of different metabolic phenotypes of P. notoginseng was elucidated. The currently proposed UPLC-ESI-MS/MS analytical method coupled with multivariate statistical analysis can be further utilized to evaluate chemical components obtained from different parts of the plant and/or the plant of different geographical locations, thereby classifying the medicinal plant resources and potentially elucidating the mechanism of inherent phytochemical diversity.

Metabonomic evaluation of melamine-induced acute renal toxicity in rats

The recent outbreak of renal failure in infants in China has been determined to be caused by melamine (Mel) and derivatives adulterated in the food. A metabonomic study was performed to evaluate the global biochemical alteration triggered by Mel ingestion in parallel with the acute renal toxicity in rats. Mel at 600, 300, and 100 mg/kg, cyanuric acid (Cya) at 100 mg/kg, and mixture of Mel and Cya (50 mg/kg each) were administered in five groups of Wistar rats, respectively, via oral gavage for 15 days. Urinary metabonomic profiles indicated that Mel perturbed urinary metabolism in a dose-dependent manner, with high-dose group showing the most significant impact. Metabonomic variations also suggest that the toxicity of low-dose (50 mg/kg) Mel was greatly elevated by the presence of Cya (at 50 mg/kg), which was able to induce a significant metabolic alteration to a level equivalent to that of 600 mg/kg Mel. Histological examination and serum biochemical analysis also indicated that the low-dose Mel-Cya mixture and high-dose Mel group resulted in the greatest renal toxicity. The high-dose Mel and low-dose Mel-Cya resulted in disrupted amino acid metabolism including tryptophan, polyamine, and tyrosine metabolism, and altered TCA and gut microflora structure.