Lee Cheng Phua

Non-invasive fecal metabonomic detection of colorectal cancer

Colorectal cancer (CRC) is a major cause of mortality in many developed countries. Effective screening strategies were called for to facilitate timely detection and to promote a better clinical outcome. In this study, the role of fecal metabonomics in the non-invasive detection of CRC was investigated. Gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) was utilized for the metabolic profiling of feces obtained from 11 CRC patients and 10 healthy subjects. Concurrently, matched tumor and normal mucosae surgically excised from CRC patients were profiled. CRC patients were differentiated clearly from healthy subjects based on their fecal metabonomic profiles (orthogonal partial least squares discriminant analysis [OPLS-DA], 1 predictive and 3 Y-orthogonal components, R2X = 0.373, R2Y = 0.995, Q2 [cumulative] = 0.215). The robustness of the OPLS-DA model was demonstrated by an area of 1 under the receiver operator characteristic curve. OPLS-DA revealed fecal marker metabolites (e.g., fructose, linoleic acid, and nicotinic acid) that provided novel insights into the tumorigenesis of CRC. Interestingly, a disparate set of CRC-related metabolic aberrations occurred at the tissue level, implying the contribution of processes beyond the direct shedding of tumor cells to the fecal metabotype. In summary, this work established proof-of-principle for GC/TOFMS-based fecal metabonomic detection of CRC and offered new perspectives on the underlying mechanisms.

Global gas chromatography/time-of-flight mass spectrometry (GC/TOFMS)-based metabonomic profiling of lyophilized human feces

Gas chromatography mass spectrometry (GC/MS)-based fecal metabonomics represents a powerful systems biology approach for elucidating metabolic biomarkers of lower gastrointestinal tract (GIT) diseases. Unlike metabolic profiling of fecal water, the profiling of complete fecal material remains under-explored. Here, a gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) method was developed and validated for the global metabonomic profiling of human feces. Fecal and fecal water metabotypes were also profiled and compared. Additionally, the unclear influence of blood in stool on the fecal metabotype was investigated unprecedentedly. Eighty milligram of lyophilized feces was ultrasonicated with 1mL of methanol:water (8:2) for 30min, followed by centrifugation, drying of supernatant, oximation and trimethylsilylation for 45min. Lyophilized feces demonstrated a more comprehensive metabolic coverage than fecal water, based on the number of chromatographic peaks. Principal component analysis (PCA) indicated occult blood (1mgHb/g feces) exerted a negligible effect on the fecal metabotype. Conversely, a unique metabotype related to feces spiked with gross blood (100mgHb/g feces) was revealed (PCA, R(2)X=0.837, Q(2)=0.794), confirming the potential confounding effect of gross GIT bleeding on the fecal metabotype. This pertinent finding highlights the importance of prudent interpretation of fecal metabonomic data, particularly in GIT diseases where bleeding is prevalent.

Global fecal microRNA profiling in the identification of biomarkers for colorectal cancer screening among Asians

Fecal microRNAs (miRNAs) are increasingly explored as non-invasive markers of colorectal cancer (CRC). However, its holistic profile in Asian CRC patients remains elusive. In the present study, the global human fecal miRNAs in Asian Chinese CRC patients was assayed to elucidate novel diagnostic fecal markers. Additionally, the influence of blood in stool on fecal miRNA levels was investigated for the first time. Microarray analysis was applied to profile the fecal miRNAs extracted from CRC patients and healthy subjects. Concurrently, surgically resected tumor and matched normal mucosae were analyzed. Potential fecal miRNA markers were further confirmed using real-time PCR in 17 CRC patients and 28 healthy subjects. Global miRNA profiling uncovered 17 fecal markers (p<0.05) differentially regulated in CRC. Fecal miR-223 and miR-451 represented robust markers in distinguishing CRC patients from healthy subjects, as evident from areas under the receiver operator characteristic curves of 0.939 and 0.971, respectively. Blood in stool affected fecal miR-451, miR-223 and miR-135b levels to a varying extent and substantially impacted the interpretation of the clinical data. Notably, a discrete set of aberrant miRNAs occurred within the tumor, indicating the presence of contributors beyond the exfoliation of tumor cells to the fecal miRNA profile. In summary, the utility of a global miRNA screening approach was successfully demonstrated in elucidating diagnostic markers of CRC. In particular, fecal miR-223 and miR-451 hold promise in detecting CRC.

Gastrointestinal Symptoms and Altered Intestinal Permeability Induced by Combat Training Are Associated with Distinct Metabotypic Changes

Physical and psychological stress have been shown to modulate multiple aspects of gastrointestinal (GI) physiology, but its molecular basis remains elusive. We therefore characterized the stress-induced metabolic phenotype (metabotype) in soldiers during high-intensity combat training and correlated the metabotype with changes in GI symptoms and permeability. In a prospective, longitudinal study, urinary metabotyping was conducted on 38 male healthy soldiers during combat training and a rest period using gas chromatography-mass spectrometry. The urinary metabotype during combat training was clearly distinct from the rest period (partial least-squares discriminant analysis (PLSDA) Q(2) = 0.581), confirming the presence of a unique stress-induced metabotype. Differential metabolites related to combat stress were further uncovered, including elevated pyroglutamate and fructose, and reduced gut microbial metabolites, namely, hippurate and m-hydroxyphenylacetate (p < 0.05). The extent of pyroglutamate upregulation exhibited a positive correlation with an increase in IBS-SSS in soldiers during combat training (r = 0.5, p < 0.05). Additionally, the rise in fructose levels was positively correlated with an increase in intestinal permeability (r = 0.6, p < 0.005). In summary, protracted and mixed psychological and physical combat-training stress yielded unique metabolic changes that corresponded with the incidence and severity of GI symptoms and alteration in intestinal permeability. Our study provided novel molecular insights into stress-induced GI perturbations, which could be exploited for future biomarker research or development of therapeutic strategies.

Investigation of the Drug-Drug Interaction Between α-Lipoic Acid and Valproate via Mitochondrial β-oxidation

ABSTRACTPurposeTo investigate the potential drug–drug interaction (DDI) between lipoic acid (LA) and valproate (VA) via the mitochondrial β-oxidation pathway in rats.MethodsIn vitro mitochondrial assays were performed to compare the biotransformation of VA to valproyl-CoA (VA-CoA), in the absence and presence of LA. In vitro microsomal and protein binding assays were performed to elucidate their potential DDI at the microsomal metabolism and distribution levels. A pharmacokinetic study was conducted in Lister Hooded rats to ascertain the in vivo DDI between LA and VA.ResultsLA was shown to decrease significantly (p < 0.05) the in vitro formation of VA-CoA in a concentration-dependent manner. Our in vitro assay results confirmed that there was minimal interaction between LA and VA in microsomal metabolism and protein binding. Based on the pharmacokinetic data, the absolute bioavailability of VA was determined to be 1.3 in the presence of LA.ConclusionsOur study demonstrated for the first time that there is a potential DDI between LA and VA at the mitochondrial β-oxidation level. While further clinical study is essential, our preliminary finding suggested that medical practitioners need to be prudent when managing epileptic patients who are co-administered with both VA and LA.

Metabolomic prediction of treatment outcome in pancreatic ductal adenocarcinoma patients receiving gemcitabine

PurposeResistance to gemcitabine remains a key challenge in the treatment of pancreatic ductal adenocarcinoma (PDAC), necessitating the constant search for effective strategies for a priori prediction of clinical outcome. While the existing studies focused on aberration of drug disposition genes and proteins as molecular predictors of gemcitabine treatment outcomes, the metabolic aberration associated with chemoresistance in clinical PDAC has been neglected. This exploratory study investigated the potential role of tissue metabolomics in characterizing the clinical treatment outcome of gemcitabine therapy.MethodsSurgically resected tumors from PDAC patients who underwent gemcitabine-based adjuvant chemotherapy (n = 25) were subjected to metabotyping using gas chromatography/time-of-flight mass spectrometry (GC/TOFMS).ResultsA partial least-squares discriminant analysis (PLS-DA) model clearly distinguished patients who had favorable survival [overall survival (OS) > 24 months] from those who exhibited poorer survival (OS < 16 months) (Q2 = 0.302). Receiver-operating characteristic analysis demonstrated the robustness of the PLS-DA model with an area under the curve of 1. PLS-DA revealed 19 marker metabolites (e.g., lactic acid, proline, and pyroglutamate) that shed insights into the chemoresistance of gemcitabine in PDAC. Particularly, tissue levels of lactic acid complemented transcript expression levels of human equilibrative nucleoside transporter 1 in distinguishing patients according to their overall survival.ConclusionThis work established proof-of-principle for GC/TOFMS-based global metabotyping of PDAC and laid the foundation for future discovery of metabolic biomarkers predictive of gemcitabine resistance in PDAC chemotherapy.

75 In Combat Soldiers Resting Urinary Metabotypes Correlate With the Development of Gastrointestinal Symptoms During Stress

Introduction: Western-diet (WD) is associated with reduced stool frequency in humans. High-fat fed mice exhibit delayed gastrointestinal (GI) transit associated with reduced number of nitrergic myenteric neurons. Mechanisms of these alterations are still unknown. Our aim was to investigate if WD feeding leads to modifications of fecal metabolites associated with the development of enteric neurodegeneration and the role played by the gut microbiota in such phenomenon. Methods: C57Bl/6 mice were fed a WD (34.5% kcal from fat, representative of the typical American diet fat calorie intake, n=5) or a Regular diet (RD, 16.9% kcal from fat, n=5). 6 weeks post feeding, fecal metabolites were analyzed by high-resolution mass spectrometry (LTQ-FT). 12 weeks post feeding, nitrergic myenteric neurons were quantified in the proximal colon by NADPH-diaphorase staining and GI and colonic transit were measured. Citrulline was measured by ELISA in germ-free (GF) mice stools fed a WD or a RD for 6 weeks (n=3 per group). Data are presentedas mean ± SEM and are significant to p < 0.05. Results: After 12 weeks of feeding, WD mice exhibited a reduced stool frequency in comparison to RD mice (1.5±0.5 and 4.4±0.3 stools/min, respectively) and this was associated with a delayed intestinal transit time (281.5±57.5 and 152.8±15.7 min). In addition, WD mice had a longer colonic transit time (as measured by bead expulsion time: WD: 127.5±52.5 min; RD: 13.2±2.0 min) associated with reduced number of nitrergic myenteric neurons in the proximal colon (113.0±5.6 and 64.2±7.4 neurons/field). Together, these data demonstrate an altered colonic motor function after 12 weeks of WD. We quantified 3730 metabolites in the feces but only 185 were significantly higher in WD mice compared to RD mice. In addition to palmitate, we found increased concentrations of citrulline (associated with enhanced nitric oxide synthase activity), 3-hydroxykynurenine (reflecting neuroinflammation) and bacterial muramic acid [Figure 1]. In order to understand the possible role of the gut microbiota in these changes, we measured fecal citrulline in GF mice fed a WD or a RD for 6 weeks and found similar concentrations (3.0±0.5 and 2.6±0.3 ng/mg). Conclusion: Our results show that long-term WD consumption is associated with nitrergic myenteric neuronal loss contributing to delayed GI transit. These alterations were preceded by gut dysbiosis and elevated fecal citrulline, potentially reflecting higher plasma concentrations that lead to oxidative stress and apoptosis in NO producing neurons. Importantly, fecal citrulline was not affected by a WD feeding under germfree conditions, suggesting that the gut microbiota is required for such effects. Fecal citrulline may be considered as a marker of myenteric neuronal impairments responsible for the motor alterations observed during WD feeding.