Eric Chun Yong Chan

Metabolic Profiling of Human Colorectal Cancer Using High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance (HR-MAS NMR) Spectroscopy and Gas Chromatography Mass Spectrometry (GC/MS)

Current clinical strategy for staging and prognostication of colorectal cancer (CRC) relies mainly upon the TNM or Duke system. This clinicopathological stage is a crude prognostic guide because it reflects in part the delay in diagnosis in the case of an advanced cancer and gives little insight into the biological characteristics of the tumor. We hypothesized that global metabolic profiling (metabonomics/metabolomics) of colon mucosae would define metabolic signatures that not only discriminate malignant from normal mucosae, but also could distinguish the anatomical and clinicopathological characteristics of CRC. We applied both high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) and gas chromatography mass spectrometry (GC/MS) to analyze metabolites in biopsied colorectal tumors and their matched normal mucosae obtained from 31 CRC patients. Orthogonal partial least-squares discriminant analysis (OPLS-DA) models generated from metabolic profiles obtained by both analytical approaches could robustly discriminate normal from malignant samples (Q(2) > 0.50, Receiver Operator Characteristic (ROC) AUC >0.95, using 7-fold cross validation). A total of 31 marker metabolites were identified using the two analytical platforms. The majority of these metabolites were associated with expected metabolic perturbations in CRC including elevated tissue hypoxia, glycolysis, nucleotide biosynthesis, lipid metabolism, inflammation and steroid metabolism. OPLS-DA models showed that the metabolite profiles obtained via HR-MAS NMR could further differentiate colon from rectal cancers (Q(2)> 0.60, ROC AUC = 1.00, using 7-fold cross validation). These data suggest that metabolic profiling of CRC mucosae could provide new phenotypic biomarkers for CRC management.

Gas chromatography/mass spectrometry in metabolic profiling of biological fluids

One of the objectives of metabonomics is to identify subtle changes in metabolite profiles between biological systems of different physiological or pathological states. Gas chromatography mass spectrometry (GC/MS) is a widely used analytical tool for metabolic profiling in various biofluids, such as urine and blood due to its high sensitivity, peak resolution and reproducibility. The availability of the GC/MS electron impact (EI) spectral library further facilitates the identification of diagnostic biomarkers and aids the subsequent mechanistic elucidation of the biological or pathological variations. With the advent of new comprehensive two dimensional GC (GC x GC) coupled to time-of-flight mass spectrometry (TOFMS), it is possible to detect more than 1200 compounds in a single analytical run. In this review, we discuss the applications of GC/MS in the metabolic profiling of urine and blood, and discuss its advances in methodologies and technologies.

Global urinary metabolic profiling procedures using gas chromatography-mass spectrometry

The role of urinary metabolic profiling in systems biology research is expanding. This is because of the use of this technology for clinical diagnostic and mechanistic studies and for the development of new personalized health care and molecular epidemiology (population) studies. The methodologies commonly used for metabolic profiling are NMR spectroscopy, liquid chromatography mass spectrometry (LC/MS) and gas chromatography–mass spectrometry (GC/MS). In this protocol, we describe urine collection and storage, GC/MS and data preprocessing methods, chemometric data analysis and urinary marker metabolite identification. Results obtained using GC/MS are complementary to NMR and LC/MS. Sample preparation for GC/MS analysis involves the depletion of urea via treatment with urease, protein precipitation with methanol, and trimethylsilyl derivatization. The protocol described here facilitates the metabolic profiling of ∼400–600 metabolites in 120 urine samples per week.

Ultra-high performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOFMS) for time-dependent profiling of raw and steamed Panax notoginseng

The metabolic profiles of Panax notoginseng and its associated therapeutic values are critically affected by the duration of steaming. The time-dependent steaming effect of P. notoginseng is not well-characterized and there is also no official guideline on its duration of steaming. In this paper, a UHPLC/TOFMS-based metabolomic platform was developed for the qualitative profiling of multiparametric metabolic changes of raw P. notoginseng during the steaming process. Our method was successful in discriminating the differentially processed herbs. Both the unsupervised principal component analysis (PCA) score plot (R(2)X=0.664, Q(2) (cum)=0.622, and PCs=2) and the supervised partial least square-data analysis (PLS-DA) model (R(2)X=0.708, R(2)Y=0.461, and Q(2)Y=0.271) demonstrated strong classification and clear trajectory patterns with regard to the duration of steaming. The PLS-DA model was validated for its robustness via a prediction set, confirming that the UHPLC/TOFMS metabolic profiles of the raw and differentially steamed P. notoginseng samples were highly reproducible. Based on our method, the minimum durations of steaming for the maximum production of bioactive ginsenosides such as Rg3 and Rh2 were also predicted. Our novel time-dependent metabolic profiling approach represents the paradigm shift in the quality control of P. notoginseng products.

Underlying mitochondrial dysfunction triggers flutamide-induced oxidative liver injury in a mouse model of idiosyncratic drug toxicity.

Flutamide, a widely used nonsteroidal anti-androgen, but not its bioisostere bicalutamide, has been associated with idiosyncratic drug-induced liver injury. Although the susceptibility factors are unknown, mitochondrial injury has emerged as a putative hazard of flutamide. To explore the role of mitochondrial sensitization in flutamide hepatotoxicity, we determined the effects of superimposed drug stress in a murine model of underlying mitochondrial abnormalities. Male wild-type or heterozygous Sod2(+/-) mice were injected intraperitoneously with flutamide (0, 30 or 100 mg/kg/day) for 28 days. A kinetic pilot study revealed that flutamide (100 mg/kg/day) caused approximately 10-fold greater exposure than the reported therapeutic mean plasma levels. Mutant (5/10), but not wild-type, mice in the high-dose group exhibited small foci of hepatocellular necrosis and an increased number of apoptotic hepatocytes. Hepatic GSSG/GSH, protein carbonyl levels, and serum lactate levels were significantly increased, suggesting oxidant stress and mitochondrial dysfunction. Measurement of mitochondrial superoxide in cultured hepatocytes demonstrated that mitochondria were a significant source of flutamide-enhanced oxidant stress. Indeed, mitochondria isolated from flutamide-treated Sod2(+/-) mice exhibited decreased aconitase activity as compared to vehicle controls. A transcriptomics analysis using MitoChips revealed that flutamide-treated Sod2(+/-) mice exhibited a selective decrease in the expression of all complexes I and III subunits encoded by mitochondrial DNA. In contrast, Sod2(+/-) mice receiving bicalutamide (50 mg/kg/day) did not reveal any hepatic changes. These results are compatible with our concept that flutamide targets hepatic mitochondria and exerts oxidant stress that can lead to overt hepatic injury in the presence of an underlying mitochondrial abnormality.

Use of urine volatile organic compounds to discriminate tuberculosis patients from healthy subjects.

Development of noninvasive methods for tuberculosis (TB) diagnosis, with the potential to be administered in field situations, remains as an unmet challenge. A wide array of molecules are present in urine and reflect the pathophysiological condition of a subject. With infection, an alteration in the molecular constituents is anticipated, characterization of which may form a basis for TB diagnosis. In the present study volatile organic compounds (VOCs) in human urine derived from TB patients and healthy controls were identified and quantified using headspace gas chromatography/mass spectrometry (GC/MS). We found significant (p < 0.05) increase in the abundance of o-xylene (6.37) and isopropyl acetate (2.07) and decreased level of 3-pentanol (0.59), dimethylstyrene (0.37), and cymol (0.42) in TB patients compared to controls. These markers could discriminate TB from healthy controls and related diseases like lung cancer and chronic obstructive pulmonary disorder. This study suggests a possibility of using urinary VOCs for the diagnosis of human TB.

Mechanism-Based Inactivation of Cytochrome P450 3A4 by Lapatinib

Fatalities stemming from hepatotoxicity associated with the clinical use of lapatinib (Tykerb), an oral dual tyrosine kinase inhibitor (ErbB-1 and ErbB-2) used in the treatment of metastatic breast cancer, have been reported. We investigated the inhibition of CYP3A4 by lapatinib as a possible cause of its idiosyncratic toxicity. Inhibition of CYP3A4 was time-, concentration-, and NADPH-dependent, with kinact = 0.0202 min−1 and Ki = 1.709 μM. The partition ratio was approximately 50.9. Addition of GSH did not affect the rate of inactivation. Testosterone protected CYP3A4 from inactivation by lapatinib. The characteristic Soret peak associated with a metabolite-intermediate complex was not observed for lapatinib during spectral difference scanning. However, reduced carbon monoxide (CO)-difference spectroscopy did reveal a 43% loss of the spectrally detectable CYP3A4-CO complex in the presence of lapatinib. Incubation of either lapatinib or its dealkylated metabolite with human liver microsomes in the presence of GSH resulted in the formation of a reactive metabolite (RM)-GSH adduct derived from the O-dealkylated metabolite of lapatinib. In addition, coincubation of lapatinib with ketoconazole inhibited the formation of the RM-GSH adduct. In conclusion, we demonstrated for the first time that lapatinib is a mechanism-based inactivator of CYP3A4, most likely via the formation and further oxidation of its O-dealkylated metabolite to a quinoneimine that covalently modifies the CYP3A4 apoprotein and/or heme moiety.

High-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometric method for the analysis of catecholamines and metanephrines in human urine

An assay of norepinephrine (NE), epinephrine (E), dopamine (DA), normetanephrine (NE) and metanephrine (MN) based on high-performance liquid chromatography (HPLC) in combination with atmospheric pressure chemical ionization mass spectrometry (APcI-MS) is described. The catecholamines and metanephrines were extracted from urine and aqueous samples using Bio-Rex 70 cation-exchange resin and subjected to analysis by HPLC/APcI-MS. The separation was performed on a C18 column in 50 mM ammonium formate buffer, pH 3.0, using a flow rate of 0.8 mL/min. Acetonitrile was added post-column at a flow rate of 0.2 mL/min via a post-column addition tee. The total analysis time was 6.5 min. The quantitative analysis was performed using 3,4-dihydroxybenzylamine (DHBA) as the internal standard (I.S.). Selected ion monitoring detection was applied: m/z 170 (for NE), 184 (for E and NM), 154 (for DA), 198 (for MN) and 140 (for DHBA, I.S.). The limits of quantitation were 5 ng/mL for NE, E and DA and 2.5 ng/mL for NM and MN. The recovery ranged from 75 to 83% for each analyte. The method was found to be simple and highly selective for the determination of catecholamines and metanephrines in the urine of patients suspected of pheochromocytoma.

Zebrafish for drug toxicity screening: bridging the in vitro cell-based models and in vivo mammalian models

Introduction: Over the past decade, zebrafish have been tasked to play important roles from modeling human diseases to finding cures for them. Inadvertently, these fish now find themselves swimming along the drug development pipeline. A number of studies have been conducted to see if these small fish are up to the task of drug toxicity testing, an important rite of passage along the pharmaceutical pipeline. Areas covered: This review covers the recent publications (2008 – 2010) on the state-of-the-art applications that couple advanced technologies with the unique advantages of zebrafish for drug toxicity screening. The paper looks at the several automated high-throughput platforms that have been developed for zebrafish teratogenicity, cardiotoxicity and neuro-sensory organ toxicity assays over the past 3 years as well as the important studies related to metabolism and biotransformation of selected drugs that have been initiated. This paper also reviews their mechanistic and predictive omics applications. Expert opinion: While there have been a number of developments over the past 3 years and indeed over the last 10 years, challenges and limitations still exist, which, unless overcome, will prevent zebrafish from truly reaching their full potential as a drug toxicological model. That being said, recent developments have suggested that zebrafish could play a role in bridging the gap between in vitro cell-based models and in vivo mammalian models.

Transposon mutagenesis identifies genes driving hepatocellular carcinoma in a chronic hepatitis B mouse model.

The most common risk factor for developing hepatocellular carcinoma (HCC) is chronic infection with hepatitis B virus (HBV). To better understand the evolutionary forces driving HCC, we performed a near-saturating transposon mutagenesis screen in a mouse HBV model of HCC. This screen identified 21 candidate early stage drivers and a very large number (2,860) of candidate later stage drivers that were enriched for genes that are mutated, deregulated or functioning in signaling pathways important for human HCC, with a striking 1,199 genes being linked to cellular metabolic processes. Our study provides a comprehensive overview of the genetic landscape of HCC.