Xiaojiao Zheng

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.

Serum and urine metabolite profiling reveals potential biomarkers of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common malignancy in the world with high morbidity and mortality rate. Identification of novel biomarkers in HCC remains impeded primarily because of the heterogeneity of the disease in clinical presentations as well as the pathophysiological variations derived from underlying conditions such as cirrhosis and steatohepatitis. The aim of this study is to search for potential metabolite biomarkers of human HCC using serum and urine metabolomics approach. Sera and urine samples were collected from patients with HCC (n = 82), benign liver tumor patients (n = 24), and healthy controls (n = 71). Metabolite profiling was performed by gas chromatography time-of-flight mass spectrometry and ultra performance liquid chromatography-quadrupole time of flight mass spectrometry in conjunction with univariate and multivariate statistical analyses. Forty three serum metabolites and 31 urinary metabolites were identified in HCC patients involving several key metabolic pathways such as bile acids, free fatty acids, glycolysis, urea cycle, and methionine metabolism. Differentially expressed metabolites in HCC subjects, such as bile acids, histidine, and inosine are of great statistical significance and high fold changes, which warrant further validation as potential biomarkers for HCC. However, alterations of several bile acids seem to be affected by the condition of liver cirrhosis and hepatitis. Quantitative measurement and comparison of seven bile acids among benign liver tumor patients with liver cirrhosis and hepatitis, HCC patients with liver cirrhosis and hepatitis, HCC patients without liver cirrhosis and hepatitis, and healthy controls revealed that the abnormal levels of glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, and chenodeoxycholic acid are associated with liver cirrhosis and hepatitis. HCC patients with alpha fetoprotein values lower than 20 ng/ml was successfully differentiated from healthy controls with an accuracy of 100% using a panel of metabolite markers. Our work shows that metabolomic profiling approach is a promising screening tool for the diagnosis and stratification of HCC 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.

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.

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.

Urinary Metabolite Markers of Precocious Puberty

The incidence of precocious puberty (PP, the appearance of signs of pubertal development at an abnormally early age), is rapidly rising, concurrent with changes of diet, lifestyles, and social environment. The current diagnostic methods are based on a hormone (gonadotropin-releasing hormone) stimulation test, which is costly, time-consuming, and uncomfortable for patients. The lack of molecular biomarkers to support simple laboratory tests, such as a blood or urine test, has been a long standing bottleneck in the clinical diagnosis and evaluation of PP. Here we report a metabolomic study using an ultra performance liquid chromatography-quadrupole time of flight mass spectrometry and gas chromatography-time of flight mass spectrometry. Urine metabolites from 163 individuals were profiled, and the metabolic alterations were analyzed after treatment of central precocious puberty (CPP) with triptorelin depot. A panel of biomarkers selected from >70 differentially expressed urinary metabolites by receiver operating characteristic and logistic regression analysis provided excellent predictive power with high sensitivity and specificity for PP. The altered metabolic profile of the PP patients was characterized by three major perturbed metabolic pathways: catecholamine, serotonin metabolism, and tricarboxylic acid cycle, presumably resulting from activation of the sympathetic nervous system and the hypothalamic-pituitary-gonadal axis. Treatment with triptorelin depot was able to normalize these three altered pathways. Additionally, significant changes in the urine levels of 4-hydroxyphenylacetic acid, 5-hydroxyindoleacetic acid, indoleacetic acid, 5-hydroxytryptophan, and 5-hydroxykynurenamine in the CPP group suggest that the development of CPP condition may involve an alteration in symbiotic gut microbial composition.

Melamine-Induced Renal Toxicity Is Mediated by the Gut Microbiota

Melamine is converted to cyanuric acid by the gut microbe Klebsiella, leading to melamine-cyanurate-urate coprecipitation that is associated with nephrotoxicity. An Unwitting Microbial Culprit in Melamine Toxicity? A tragic incident in China in 2008 involving the deliberate and illicit supplementation of milk with melamine resulted in the deaths of children from renal failure and highlighted the toxicity of this compound. In a new study, Zheng et al. study the toxicity of melamine in rats and report that microbial metabolism of melamine is crucial for forming the key metabolite that causes kidney damage. They discover that a species of Klebsiella can form cyanuric acid from melamine, which then forms complex precipitates that lead to kidney stone formation. The gut microbiota in general play an important role in human health and are known to affect the metabolism and toxicity of a number of drugs. The new findings suggest that individual variation in the gut microbial composition of children exposed to melamine may have been important in the observed patterns of mortality in exposed individuals. Melamine poisoning has become widely publicized after a recent occurrence of renal injury in infants and children exposed to melamine-tainted milk in China. This renal damage is believed to result from kidney stones formed from melamine and uric acid or from melamine and its cocrystallizing chemical derivative, cyanuric acid. However, the composition of the stones and the mechanism by which the stones are formed in the renal tubules are unknown. We report that cyanuric acid can be produced in the gut by microbial transformation of melamine and serves as an integral component of the kidney stones responsible for melamine-induced renal toxicity in rats. Melamine-induced toxicity in rats was attenuated and melamine excretion increased after antibiotic suppression of gut microbial activity. We further demonstrated that melamine is converted to cyanuric acid in vitro by bacteria cultured from normal rat feces; Klebsiella was subsequently identified in fecal samples by 16S ribosomal DNA sequencing. In culture, Klebsiella terrigena was shown to convert melamine to cyanuric acid directly. Rats colonized by K. terrigena showed exacerbated melamine-induced nephrotoxicity. Cyanuric acid was detected in the kidneys of rats administered melamine alone, and the concentration after Klebsiella colonization was increased. These findings suggest that the observed toxicity of melamine may be conditional on the exact composition and metabolic activities of the gut microbiota.

Alteration of bile acid metabolism in the rat induced by chronic ethanol consumption

Our understanding of the bile acid metabolism is limited by the fact that previous analyses have primarily focused on a selected few circulating bile acids; the bile acid profiles of the liver and gastrointestinal tract pools are rarely investigated. Here, we determined how chronic ethanol consumption altered the bile acids in multiple body compartments (liver, gastrointestinal tract, and serum) of rats. Rats were fed a modified Lieber‐DeCarli liquid diet with 38% of calories as ethanol (the amount equivalent of 4–5 drinks in humans). While conjugated bile acids predominated in the liver (98.3%), duodenum (97.8%), and ileum (89.7%), unconjugated bile acids comprised the largest proportion of measured bile acids in serum (81.2%), the cecum (97.7%), and the rectum (97.5%). In particular, taurine‐conjugated bile acids were significantly decreased in the liver and gastrointestinal tract of ethanol‐treated rats, while unconjugated and glycine‐conjugated species increased. Ethanol consumption caused increased expression of genes involved in bile acid biosynthesis, efflux transport, and reduced expression of genes regulating bile acid influx transport in the liver. These results provide an improved understanding of the systemic modulations of bile acid metabolism in mammals through the gut‐liver axis.—Xie, G., Zhong, W., Li, H., Li, Q., Qiu, Y., Zheng, X., Chen, H., Zhao, X., Zhang, S., Zhou, Z., Zeisel, S. H., Jia, W., Alteration of bile acid metabolism in the rat induced by chronic ethanol consumption. FASEB J. 27, 3583–3593 (2013). www.fasebj.org

Chronic ethanol consumption alters mammalian gastrointestinal content metabolites.

Chronic ethanol consumption is associated with not only the alteration of metabolic profiles in biofluids but also the composition of the gut microbiome. Our understanding of the importance of the intestinal microbiota as well as the disturbances elicited by ethanol intervention is limited by the fact that previous analyses have primarily focused on biofluids and liver tissue metabolome; the metabolic profiles of the gastrointestinal (GI) contents are rarely investigated. In this study, we applied a metabonomics approach using a high performance liquid chromatography-time-of-flight mass spectrometry (HPLC-TOF MS) and gas chromatography-mass spectrometry (GC-MS) to characterize the metabolic alterations of the contents within the GI tract (stomach, duodenum, jejunum, ileum, cecum, colon, and rectum) in male Sprague-Dawley rats following 8 weeks of ethanol exposure. We obtained a snapshot of the distinct changes of the intestinal content metabolite composition in rats with ethanol exposure, which indicated a profound impact of ethanol consumption on the intestinal metabolome. Many metabolic pathways that are critical for host physiology were affected, including markedly altered bile acids, increased fatty acids and steroids, decreased carnitines and metabolites involved in lipid metabolism, a significant decrease of all amino acids and branched chain amino acids, and significantly decreased short chain fatty acids except for acetic acid, which rapidly elevated as a product of ethanol metabolism. These results provide an improved understanding of the systemic alteration of intestinal content metabolites in mammals and the interplay between the host and its complex resident microbiota and may aid in the design of new therapeutic strategies that target these interactions.

Metabolomics approaches for characterizing metabolic interactions between host and its commensal microbes

It is increasingly evident that the gut microbiota is involved in the regulation of multiple mammalian metabolic pathways through a series of interactive host‐microbiota metabolic, signaling, and immune‐inflammatory axes that physiologically connect the gut, liver, brain, and other organs. Correlation of the metabotypes with the gut microbial profiles derived from culture‐independent molecular techniques is increasingly useful for deciphering inherent and intimate host–microbe relationships. Real‐time analysis of the small molecule metabolites derived from gut microbial‐host co‐metabolism is essential for understanding the metabolic functions of the gut microbiome and has tremendous implications for personalized healthcare strategies. Metabolomics, an array of analytical techniques that includes high resolution NMR spectroscopy and chromatography‐MS in conjunction with chemometrics and bioinformatics tools, enables characterization of the metabolic footprints of mammalian hosts that correlate with the microbial community in the intestinal tract. The metabolomics approach provides important information of a complete spectrum of metabolites produced from the gut microbial‐mammalian co‐metabolism and is improving our understanding of the molecular mechanisms underlying multilevel host–microbe interactions. In this review, the interactions of gut microbiota with their host are discussed and some examples of NMR‐ or MS‐based metabolomics applications for characterizing the metabolic footprints of gut microbial‐host co‐metabolism are described. Advances in the metabolomic analysis of bile acids, short‐chain fatty acids, and choline metabolism are also summarized.