Shuhai Lin

GC/MS-based metabolomics reveals fatty acid biosynthesis and cholesterol metabolism in cell lines infected with influenza A virus

Metabolomics is the downstream of systems biology and has drawn significant interest for studying the metabolic networks from cells to organisms. To profile the metabolites in two different cell lines (A549 and AGS) infected with influenza A virus, gas chromatography coupled with mass spectrometry (GC/MS) was employed. Some differentiating metabolites in the cell lines were tentatively identified using reference library, interpreted and visualized by applying principal components analysis (PCA) and cluster heat map. Consequently, metabolic flux profiling allowed the differentiation of fatty acid biosynthesis and cholesterol metabolism during viral replication in the cell lines. The change in fatty acid turnover was also observed. Metabolomics investigation also revealed the different responses between A549 and AGS cell lines to the virus infection. From the pattern recognition results, AGS cell line might be more susceptible to influenza A virus. Regarding the fact that AGS is a poorly differentiated gastric adenocarcinoma cell line whereas A549 is a relatively differentiated lung tumor one, it is speculated that viral replication might be associated with the cell differentiations.

Acetate functions as an epigenetic metabolite to promote lipid synthesis under hypoxia

Besides the conventional carbon sources, acetyl-CoA has recently been shown to be generated from acetate in various types of cancers, where it promotes lipid synthesis and tumour growth. The underlying mechanism, however, remains largely unknown. We find that acetate induces a hyperacetylated state of histone H3 in hypoxic cells. Acetate predominately activates lipogenic genes ACACA and FASN expression by increasing H3K9, H3K27 and H3K56 acetylation levels at their promoter regions, thus enhancing de novo lipid synthesis, which combines with its function as the metabolic precursor for fatty acid synthesis. Acetyl-CoA synthetases (ACSS1, ACSS2) are involved in this acetate-mediated epigenetic regulation. More importantly, human hepatocellular carcinoma with high ACSS1/2 expression exhibit increased histone H3 acetylation and FASN expression. Taken together, this study demonstrates that acetate, in addition to its ability to induce fatty acid synthesis as an immediate metabolic precursor, also functions as an epigenetic metabolite to promote cancer cell survival under hypoxic stress.

Pregnancy-Induced Metabolic Phenotype Variations in Maternal Plasma

Metabolic variations occur during normal pregnancy to provide the growing fetus with a supply of nutrients required for its development and to ensure the health of the woman during gestation. Mass spectrometry-based metabolomics was employed to study the metabolic phenotype variations in the maternal plasma that are induced by pregnancy in each of its three trimesters. Nontargeted metabolomics analysis showed that pregnancy significantly altered the profile of metabolites in maternal plasma. The levels of six metabolites were found to change significantly throughout pregnancy, with related metabolic pathway variations observed in biopterin metabolism, phospholipid metabolism, amino acid derivatives, and fatty acid oxidation. In particular, there was a pronounced elevation of dihydrobiopterin (BH₂), a compound produced in the synthesis of dopa, dopamine, norepinephrine, and epinephrine, in the second trimester, whereas it was markedly decreased in the third trimester. The turnover of BH₂ and tryptophan catabolites indicated that the fluctuations of neurotransmitters throughout pregnancy might reveal the metabolic adaption in the maternal body for the growth of the fetus. Furthermore, 11 lipid classes and 41 carnitine species were also determined and this showed variations in the presence of long-chain acylcarnitines and lysophospholipids in later pregnancy, suggesting changes of acylcarnitines and lysophospholipids to meet the energy demands in pregnant women. To our knowledge, this work is the first report of dynamic metabolic signatures and proposed related metabolic pathways in the maternal plasma for normal pregnancies and provided the basis for time-dependent metabolic trajectory against which disease-related disorders may be contrasted.

Metabolomics study of alcohol-induced liver injury and hepatocellular carcinoma xenografts in mice

Alcohol abuse is one of the major causes of liver injury and a promoter for hepatocellular carcinoma (HCC). To understand the disease-associated metabolic changes, we investigated and compared the profiles of metabolites in nude mice with alcohol-induced liver injury or bearing a HCC xenograft (HCCX). Alcohol-induced liver injury was achieved by daily administration of grain liquor, and HCC xenografts were generated by subcutaneous inoculation of HepG2 cells in nude mice. Metabolites in serum samples were profiled by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS). The acquired data was analyzed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) to identify potential disease-specific biomarkers. Results showed that the phosphatidylcholine (PC) levels were significantly higher in both liver injury and HCCX mice compared with the control. Interestingly, lysophosphatidylcholines (LPCs) that contain saturated or monounsaturated fatty acids were reduced in both liver injury and HCCX mice, but polyunsaturated fatty acids LPCs were elevated in liver injury mice only. These data delineated the disease-related metabolic alterations of LPCs in liver injury and HCC, suggesting that the LPC profile in serum may be biomarkers for these two common liver diseases.

Ultrahigh resolution mass spectrometry-based metabolic characterization reveals cerebellum as a disturbed region in two animal models.

In the previous reports about cognitive dysfunction, cerebellum was thought to be a less affected tissue by genetic or environmental alterations in comparison to other tissues in the brain including hippocampus under the same conditions. In this work, we investigated two types of metabolomic alterations inside the cerebellum tissue. The first one addressed the differences in the metabolomics profiles between Transgenic (Tg) CRND8 of Alzheimers disease mice and non-transgenic (non-Tg) littermates. The second one addressed the metabolic differences between wild type mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and wild type mice which are not exposed to this toxic compound. For these two investigations, ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS) was implemented. As a result, the significant changes of each comparison were tentatively annotated by the high mass accuracy generated from the measurements in the negative ion mode. The biosynthesis of amino acids was also enhanced pronouncedly, and perturbation of purine metabolism was also observed in Tg mice compared to non-Tg littermates. In another animal model, the reduced levels of amino acids were found whereas the intermediate levels in purine metabolism and fatty acids including fatty acid conjugated metabolites were elevated in cerebellar tissues of mice exposed to TCDD compared to control group. Collectively, it was demonstrated that FT-ICR/MS was a powerful tool for interpretation of the elemental compositions of the peaks, revealing that the metabolic perturbations in cerebellar tissues of mice were induced by either genetic manipulation or environmental factor. Therefore, the non-targeted approach, alternatively, provides various metabolic phenotypes for the systems-level mirror of the complex etiology of neurotoxicity in the cerebellum.

Metabolite profiling of plasma and urine from rats with TNBS-induced acute colitis using UPLC-ESI-QTOF-MS-based metabonomics – a pilot study

The incidence of inflammatory bowel disease, a relapsing intestinal condition whose precise etiology is still unclear, has continually increased over recent years. Metabolic profiling is an effective method with high sample throughput that can detect and identify potential biomarkers, and thus may be useful in investigating the pathogenesis of inflammatory bowel disease. In this study, using a metabonomics approach, a pilot study based on ultra‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry (UPLC‐ESI‐QTOF‐MS) was performed to characterize the metabolic profile of plasma and urine samples of rats with experimental colitis induced by 2,4,6‐trinitrobenzene sulfonic acid. Acquired metabolic profile data were processed by multivariate data analysis for differentiation and screening of potential biomarkers. Five metabolites were identified in urine: two tryptophan metabolites [4‐(2‐aminophenyl)‐2,4‐dioxobutanoic acid and 4,6‐cihydroxyquinoline], two gut microbial metabolites (phenyl‐acetylglycine and p‐cresol glucuronide), and the bile acid 12α‐hydroxy‐3‐oxocholadienic acid. Seven metabolites were identified in plasma: three members of the bile acid/alcohol group (cholic acid, 12α‐hydroxy‐3‐oxocholadienic acid and cholestane‐3,7,12,24,25‐pentol) and four lysophosphatidylcholines [LysoPC(20:4), LysoPC(16:0), LysoPC(18:1) and LysoPC(18:0)]. These metabolites are associated with damage of the intestinal barrier function, microbiota homeostasis, immune modulation and the inflammatory response, and play important roles in the pathogenesis of inflammatory bowel disease. Our results positively support application of the metabonomic approach in study of the pathophysiological mechanism of inflammatory bowel disease.

Liquid chromatography/mass spectrometry for investigating the biochemical effects induced by aristolochic acid in rats: the plasma metabolome.

In this study, high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry in conjunction with chemometric methods including principal components analysis was used to investigate metabolic profiling in plasma samples from rats dosed with aristolochic acid (AA). Differentiating metabolites were identified by high-resolution mass spectrometry and tandem mass spectrometry analyses, database searching and comparison with the analytical results of authentic standards. Several metabolites in plasma including a glucuronide conjugate, bile acids, lysophosphatidylcholines and fatty acids were detected in rats exposed to AA. To improve visualization, a z-score plot and a cluster heat map were generated for the concentration fluctuations of the metabolites in different dosage groups. The results obtained from this study indicated that class-specific metabolomic patterns were obviously differentiated for each metabolite in the different dosage groups of AA.

Removal and reductive dechlorination of triclosan by Chlorella pyrenoidosa.

Triclosan that is widely used as antimicrobial agent has been detected as contaminant in various aquatic environments. In this work, removal and biodegradation of triclosan in water by using a ubiquitous green alga, Chlorella pyrenoidosa was investigated. When C. pyrenoidosa was exposed to a series concentration of triclosan from 100 to 800ngmL(-1), more than 50% of triclosan was eliminated by algal uptake from the culture medium during the first 1h exposure and reached equilibrium after the 6h treatment. In the biodegradation experiments, a removal percentage of 77.2% was obtained after C. pyrenoidosa was cultivated with 800ngmL(-1) triclosan for 96h. A major metabolite from the reductive dechlorination of triclosan was identified by using liquid chromatography coupled with electrospray ionization-mass spectrometry. The ultrastructural morphology of algal cells grown in the presence of triclosan was observed by using transmission electron microscopy and the growth of algal cells was detected. It was found that the trilcosan treatment resulted in the disruption of the chloroplast and the release of organic material into aquatic environment, which indicated that triclosan may affect membrane metabolism.

A novel method of liquid chromatography-tandem mass spectrometry combined with chemical derivatization for the determination of ribonucleosides in urine

Ribonucleosides are the end products of RNA metabolism. These metabolites, especially the modified ribonucleosides, have been extensively evaluated as cancer-related biomarkers. However, the determination of urinary ribonucleosides is still a challenge due to their low abundance, high polarity and serious matrix interferences in urine samples. In this study, a derivatization method based on a chemical reaction between ribonucleosides and acetone to form acetonides was developed for the determination of urinary ribonucleosides. The derivative products, acetonides, were detected by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The methodological evaluation was performed by quantifying four nucleosides for linear range, average recovery, precision, accuracy and stability. The validated procedures were applied to screen modified ribonucleosides in urine samples. Improvement of separation and enhancement of sensitivity were obtained in the analysis. To identify ribonucleosides, inexpensive isotope labeling acetone (acetone-d6) and label-free acetone were applied to form ordinary and deuterated acetonides, respectively. The two groups of samples were separated with orthogonal partial least squares (OPLS). The ordinary and deuterated pairs of acetonides were symmetrically distributed in the S-plot for easy and visual signal identification. After structural confirmation, a total of 56 ribonucleosides were detected, 52 of which were modified ribonucleosides. The application of derivatization, deuterium-labeling and multivariate statistical analysis offers a new option for selective detection of ribonucleosides in biological samples.

Simultaneous determination of ten compounds in rat plasma by UPLC-MS/MS: Application in the pharmacokinetic study of Ma-Zi-Ren-Wan

Ma-Zi-Ren-Wan (MZRW) is a classic Chinese formula which has been used to treat human constipation in China for over 2000 years. In order to make good and rational use of this formula in the future, this paper presents the first attempt to track the pharmacokinetic features of MZRW in rat using rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Ten chemical components of MZRW, namely, rhein, emodin, aloe emodin, hesperidin, naringin, amygdalin, albiflorin, paeoniflorin, magnolol and honokiol, were simultaneously determined in rat plasma after a single oral administration (10g/kg body weight) of MZRW to rats. Geniposide and liquiritin were used as internal standards. The separation was performed on a Waters ACQUITY BEH C18 column (100mm×2.1mm, 1.7μm). The detection was conducted by multiple-reaction monitoring (MRM) in negative ionization mode. Two highest abundant MRM transitions without interference were optimized for each analyte. This method was well validated in terms of linearity, precision, accuracy, recovery, matrix effect and stability. All calibration curves had good linearity (r(2)>0.995) over the concentration range from 3.9 to 125.0ng/mL for emodin, 3.9-500.0ng/mL for amygdalin, 2.0-4000.0ng/mL for naringin and hesperidin, 3.9-2000.0ng/mL for magnolol, 7.8-2000.0ng/mL for rhein and 3.9-4000.0ng/mL for albiflorin, paeoniflorin, aloe emodin and honokiol. The intra-day and inter-day precision (relative standard deviation) was within 15%, the accuracy (relative error) ranged from -13.6% to 15.1%, and the lower limit of quantification in plasma ranged between 2.0ng/mL and 7.8ng/mL. Extraction recovery, matrix effect and stability were satisfactory. The validated method was successfully applied to a pharmacokinetic study of these ten compounds after oral administration of MZRW to rats. The pharmacokinetic parameters of each compound can facilitate clinical studies in the future.