Metabotyping of human colorectal cancer using two-dimensional gas chromatography mass spectrometry.

Colorectal cancer (CRC) is the fourth most common cause of death from cancer in the world. The limitations of the currently available methods and biomarkers for CRC management highlight the necessity of finding novel markers. Metabonomics can be used to search for potential markers that can provide molecular insight into human CRC. The emergence of two-dimensional gas chromatography time of flight mass spectrometry (GC × GC/TOFMS) has comprehensively enhanced the metabolic space coverage of conventional GC/MS. In this study, a GC × GC/TOFMS was developed for the tissue-based global metabonomic profiling of CRC. A Pegasus GC × GC/TOFMS (Leco Corp., St. Joseph, MI, USA) system comprising an Agilent 7890 GC and Pegasus IV TOFMS was used for this purpose. An Agilent DB-1 (30 m × 250 µm × 0.25 µm) fused silica capillary column and a Restek Rxi®-17 (1 m × 100 µm × 0.10 µm) fused silica capillary column were used as the primary and secondary columns, respectively. The method was applied for global metabonomic profiling of matched CRC and normal tissues (n = 63) obtained from 31 CRC patients during surgery. An attempt was also made to compare GC × GC/TOFMS with GC/MS and NMR in similar application. The results showed that the metabotype associated with CRC is distinct from that of normal tissue and led to the identification of chemically diverse marker metabolites. Metabolic pathway mapping suggested deregulation of various biochemical processes such as glycolysis, Krebs cycle, osmoregulation, steroid biosynthesis, eicosanoid biosynthesis, bile acid biosynthesis, lipid, amino acid and nucleotide metabolism.

Metabolic profiling of 3-nitropropionic acid early-stage Huntington's disease rat model using gas chromatography time-of-flight mass spectrometry.

3-Nitropropionic acid (3-NP), a potent irreversible inhibitor of mitochondrial complex II enzyme, leads to mitochondrial dysfunction and oxidative stress in Huntington's disease (HD) rat model. In this study, biochemical assays were used to demonstrate the presence of oxidative stress and mitochondrial dysfunction in 3-NP early stage HD rat models. Gas chromatography time-of-flight mass spectrometry (GC/TOFMS) was applied to analyze metabolites in brain and plasma of 3-NP-treated and vehicle-dosed rats. The orthogonal partial least-squares discriminant analysis (OPLS-DA) model generated using brain metabolic profiles robustly differentiated the 3-NP early stage HD rat model from the control. Metabonomic characterization of the 3-NP HD rat model facilitated the detection of biomarkers that define the physiopathological phenotype of early stage HD and elucidated the treatment effect of galantamine. Brain marker metabolites that were identified based on the OPLS-DA model were associated with altered glutathione metabolism, oxidative stress, and impaired energy metabolism. The treatment effect of galantamine in early stage HD could not be concluded mechanistically using the brain metabotype. Our study confirmed that GC/TOFMS is a strategic and complementary platform for the metabonomic characterization of 3-NP induced neurotoxicity in the early stage HD rat model.

Ultra-pressure liquid chromatography/tandem mass spectrometry targeted profiling of arachidonic acid and eicosanoids in human colorectal cancer.

Cumulative evidence shows that eicosanoids such as prostaglandins, leukotrienes, thromboxanes and hydroxy eicosatetraenoic acids play an important role in associating inflammation with human colorectal cancer (CRC). In this study an ultra-pressure liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed and validated for the targeted profiling of eight relevant eicosanoids and the major metabolic precursor, arachidonic acid (AA), in human colon. Multiple reaction monitoring (MRM) experiments were performed in negative electrospray ionization mode. The metabolites were separated using a C(18) column consisting of 1.7 µm ethylene-bridged hybrid particles (100 × 2.1 mm i.d.) and gradient elution (50 to 95% of solvent B) with a mobile phase comprising water (0.1% formic acid) [solvent A] and acetonitrile (0.1% formic acid) [solvent B] at a flow rate of 0.4 mL/min. The analysis time for each sample was 5.5 min. Our UPLC/MS/MS method demonstrated satisfactory validation results in terms of selectivity, sensitivity, matrix effect, linearity, extraction efficiency, intra- and inter-day precision, accuracy and autosampler stability. The method was applied for the clinical profiling of matched pairs of cancerous and normal colon mucosae obtained from eight colorectal cancer patients. Endogenous levels of AA and selected eicosanoids such as prostaglandin E(2) (PGE(2)), prostacyclin (PGI(2)) [assayed as its stable hydrolytic product 6-keto-prostaglandin(1α) (6-k PGF(1α))] and 12-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12-HETE) were found to be significantly different (p <0.05; paired t-test) between cancerous and normal mucosae.

Validation of HPTLC method for the analysis of taraxerol in Clitoria ternatea.

A new, simple, sensitive, selective and precise HPTLC method has been developed for the determination of taraxerol in Clitoria ternatea L. Determination of taraxerol was performed on TLC aluminium plates. Linear ascending development was carried out in twin trough glass chamber saturated with hexane and ethyl acetate (80:20 v/v). The plate was then dried and sprayed with anisaldehyde reagent. A Camag TLC scanner III was used for spectrodensitometric scanning and analysis at 420 nm. The system was found to give compact spots for taraxerol (R(f) 0.53). The calibration plot was linear in the range of 100-1200 ng of taraxerol. The correlation coefficient of 0.9961 was indicative of good linear dependence of peak area on concentration. The concentration of taraxerol was found to be 12.4 mg/g w/w in the hydroalcoholic extract of C. ternatea root. To study the accuracy and precision of the method, recovery studies were performed. Recovery values from 99.65 to 99.74% showed excellent reliability and reproducibility of the method. The limits of detection and quantification were determined to be 31 and 105 ng/spot, respectively. The proposed HPTLC method for quantitative monitoring of taraxerol in C. ternatea can be used for routine quality testing of C. ternatea extract used in Ayurvedic formulations.

Noninvasive metabolic profiling for painless diagnosis of human diseases and disorders.

Metabolic profiling provides a powerful diagnostic tool complementary to genomics and proteomics. The pain, discomfort and probable iatrogenic injury associated with invasive or minimally invasive diagnostic methods, render them unsuitable in terms of patient compliance and participation. Metabolic profiling of biomatrices like urine, breath, saliva, sweat and feces, which can be collected in a painless manner, could be used for noninvasive diagnosis. This review article covers the noninvasive metabolic profiling studies that have exhibited diagnostic potential for diseases and disorders. Their potential applications are evident in different forms of cancer, metabolic disorders, infectious diseases, neurodegenerative disorders, rheumatic diseases and pulmonary diseases. Large scale clinical validation of such diagnostic methods is necessary in future.

Ablation of dihydroceramide desaturase 1, a therapeutic target for the treatment of metabolic diseases, simultaneously stimulates anabolic and catabolic signaling.

The lipotoxicity hypothesis posits that obesity predisposes individuals to metabolic diseases because the oversupply of lipids to tissues not suited for fat storage leads to the accumulation of fat-derived molecules that impair tissue function. Means of combating this have been to stimulate anabolic processes to promote lipid storage or to promote catabolic ones to drive fat degradation. Herein, we demonstrate that ablating dihydroceramide desaturase 1 (Des1), an enzyme that produces ceramides, leads to the simultaneous activation of both anabolic and catabolic signaling pathways. In cells lacking Des1, the most common sphingolipids were replaced with dihydro forms lacking the double bond inserted by Des1. These cells exhibited a remarkably strong activation of the antiapoptotic and anabolic signaling pathway regulated by Akt/protein kinase B (PKB), were resistant to apoptosis, and were considerably larger than their wild-type counterparts. Paradoxically, Des1(-/-) cells exhibited high levels of autophagy. Mechanistic studies revealed that this resulted from impaired ATP synthesis due in part to decreased expression and activity of several complexes of the electron transport chain, particularly complex IV, leading to activation of AMP-activated protein kinase and its induction of the autophagosome. Thus, Des1 ablation enhanced starvation responses but dissociated them from the anabolic, prosurvival, and antiautophagic Akt/PKB pathways.

Investigating the role of nucleoside transporters in the resistance of colorectal cancer to 5-fluorouracil therapy.

Resistance to 5-fluorouracil (5FU) poses a constant challenge to the management of colorectal cancer (CRC). Consistent efforts were called for to identify molecular markers that can effectively predict patients' response. This study investigated the role of nucleoside transporters, particularly human equilibrative nucleoside transporter 1 (hENT1), in predicting clinical treatment outcome with 5FU-based therapy. Expression of a panel of nucleoside transporters in biopsied tumors from 7 CRC patients was measured by real-time PCR prior to 5FU-based chemotherapy. To provide mechanistic support for the role of hENT1 in 5FU resistance, cell viability of Caco-2 cells was measured, following incubation with varying concentrations of 5FU and a hENT1 inhibitor. Biopsied tumors were further subjected to global metabonomic profiling using gas chromatography/mass spectrometry. High hENT1 levels in tumor tissue correlated with poor clinical response to 5FU. Corroborating with the clinical findings, chemical inhibition of hENT1 in Caco-2 cells resulted in an augmentation of 5FU efficacy. Metabonomic profiling revealed that the pretreatment metabotype associated with non-responders to 5FU therapy was distinct from metabotype of responders (partial least-squares discriminant analysis Q(2) (cumulative) = 0.898, R(2)X = 0.513, R(2)Y = 0.996). This is the first clinical report on the relationships of intratumoral expression of nucleoside transporters and tumor metabotype with response to 5FU among CRC patients. Coupled to the in vitro findings, our preliminary data suggested hENT1 to be a potential codeterminant of clinical response to 5FU.

Development and validation of a gas chromatography/mass spectrometry method for the metabolic profiling of human colon tissue.

In this study, a gas chromatography/mass spectrometry (GC/MS) method was developed and validated for the metabolic profiling of human colon tissue. Each colon tissue sample (20 mg) was ultra-sonicated with 1 mL of a mixture of chloroform/methanol/water in the ratio of 20:50:20 (v/v/v), followed by centrifugation, collection of supernatant, drying, removal of moisture using anhydrous toluene and finally derivatization using N-methyl-N-trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS). A volume of 1 microL of the derivatized mixture was injected into the GC/MS system. A total of 53 endogenous metabolites were separated and identified in the GC/MS chromatogram, all of which were selected to evaluate the sample stability and precision of the method. Of the identified endogenous metabolites 19 belonging to diverse chemical classes and covering a wide range of the GC retention times (Rt) were selected to investigate the quantitative linearity of the method. The developed GC/MS method demonstrated good reproducibility with intra- and inter-day precision within relative standard deviation (RSD) of +/-15%. The metabolic profiles of the intact tissue were determined to be stable (100 +/- 15%) for up to 90 days at -80 degrees C. Satisfactory results were also obtained in the case of other stability-indicating studies such as freeze/thaw cycle stability, bench-top stability and autosampler stability. The developed method showed a good linear response for each of the 19 analytes tested (r(2) > 0.99). Our GC/MS metabolic profiling method was successfully applied to discriminate biopsied colorectal cancer (CRC) tissue from their matched normal tissue obtained from six CRC patients using orthogonal partial least-squares discriminant analysis [two latent variables, R(2)Y = 0.977 and Q(2) (cumulative) = 0.877].

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.

In vitro acetylcholinesterase inhibitory activity of the essential oil from Acorus calamus and its main constituents.

The in vitro acetylcholinesterase (AChE) inhibitory potential of the hydroalcoholic extract and of the essential oil from Acorus calamus (AC) rhizomes and that of its major constituents were evaluated based on the Ellman's method. GC/MS analysis of the oil revealed that the major constituents were beta-asarone (79.54%) and alpha-asarone (8.47%). The IC50 values were obtained for the hydroalcoholic extract, the essential oil, beta-asarone and alpha-asarone and were 182.31+/-16.78 microg/mL, 10.67+/-0.81 microg/mL, 3.33+/-0.02 microM and 46.38+/-2.69 microM, respectively. Physostigmine was used as standard inhibitor with an IC50 value of 0.28+/-0.015 microM. The experimental observations revealed that the AC essential oil and its constituents have significant AChE inhibitory potential. beta-Asarone, the major phytoconstituent present in the essential oil, showed the maximum inhibitory potential.

Acetylcholinesterase inhibitors from plants.

Inhibition of acetylcholinesterase (AChE), the key enzyme in the breakdown of acetylcholine, is considered as a promising strategy for the treatment of neurological disorders such as Alzheimer's disease, senile dementia, ataxia and myasthenia gravis. A potential source of AChE inhibitors is certainly provided by the abundance of plants in nature. This article aims to provide a comprehensive literature survey of plants that have been tested for AChE inhibitory activity. Numerous phytoconstituents and promising plant species as AChE inhibitors are being reported in this communication.

Determination of 6-gingerol in ginger (Zingiber officinale) using high-performance thin-layer chromatography.

A sensitive and accurate High-Performance TLC (HPTLC) method has been developed to determine the quantity of 6-gingerol in rhizomes of Zingiber officinale (family: Zingiberaceae), commonly known as ginger. Methanol extracts of rhizomes from three different sources were used for HPTLC, n-hexane, and diethyl ether (40:60 v/v) as the mobile phase. The Rf of 6-gingerol was found to be 0.40. The calibration plot was linear in the range of 250-1200 ng of 6-gingerol and the correlation coefficient of 0.9997 was indicative of good linear dependence of peak area on concentration. The mean quantity of 6-gingerol was found to be 60.44+/-2.53 mg/g of ginger extract. The method permits reliable quantification of 6-gingerol and good resolution and separation of 6-gingerol from other constituents of ginger. To study the accuracy and precision of the method, recovery studies were performed by the method of standard addition. Recovery values from 99.79 to 99.84% showed the excellent reliability and reproducibility of the method. The proposed HPTLC method for quantitative monitoring of 6-gingerol in ginger can be used for routine quality testing of ginger extracts.