2D 1H-13C Research Articles

Impact of ultrasounds and high voltage electrical discharges on physico-chemical properties of rapeseed straw’s lignin and pulps

In this study, ultrasound (US) and high voltage electrical discharges (HVED) were combined with chemical treatments (soda or organosolv) for rapeseed straw delignification.Delignification was improved by both physical pretreatments. US increased the extractability of hemicelluloses and HVED induced a partial degradation of cellulose. Best synergies were observed for HVED-soda and US-organosolv treatments. The obtained lignin fractions were characterized with 13C NMR and 2D 1H–13C HSQC. It was observed that the physical treatments affected the syringyl/guaiacyl (S/G) ratios. The values of S/G were ≈1.19, 1.31 and 1.75 for organosolv, HVED-organosolv and US-organosolv processes, suggesting recondensation reactions. The lignin fractions obtained from HVED-organosolv treatment contained less quantity of p-coumaric acid and ferulic acid as compared to those extracted by US-organosolv. Thermogravimetric analysis (TGA) revealed a better heat resistance of physically extracted lignins as compared to the control. The enzymatic digestibility increased by 24.92% when applying HVED to mild organosolv treatment.

Enhancing the resolution of 1H and 13C solid-state NMR spectra by reduction of anisotropic bulk magnetic susceptibility broadening.

We demonstrate that natural isotopic abundance 2D heteronuclear correlation (HETCOR) solid-state NMR spectra can be used to significantly reduce or eliminate the broadening of 1H and 13C solid-state NMR spectra of organic solids due to anisotropic bulk magnetic susceptibility (ABMS). ABMS often manifests in solids with aromatic groups, such as active pharmaceutical ingredients (APIs), and inhomogeneously broadens the NMR peaks of all nuclei in the sample. Inhomogeneous peaks with full widths at half maximum (FWHM) of ∼1 ppm typically result from ABMS broadening and the low spectral resolution impedes the analysis of solid-state NMR spectra. ABMS broadening of solid-state NMR spectra has previously been eliminated using 2D multiple-quantum correlation experiments, or by performing NMR experiments on diluted materials or single crystals. However, these experiments are often infeasible due to their poor sensitivity and/or provide limited gains in resolution. 2D 1H-13C HETCOR experiments have previously been applied to reduce susceptibility broadening in paramagnetic solids and we show that this strategy can significantly reduce ABMS broadening in diamagnetic organic solids. Comparisons of 1D solid-state NMR spectra and 1H and 13C solid-state NMR spectra obtained from 2D 1H-13C HETCOR NMR spectra show that the HETCOR spectrum directly increases resolution by a factor of 1.5 to 8. The direct gain in resolution is determined by the ratio of the inhomogeneous 13C/1H linewidth to the homogeneous 1H linewidth, with the former depending on the magnitude of the ABMS broadening and the strength of the applied field and the latter on the efficiency of homonuclear decoupling. The direct gains in resolution obtained using the 2D HETCOR experiments are better than that obtained by dilution. For solids with long proton longitudinal relaxation times, dynamic nuclear polarization (DNP) was applied to enhance sensitivity and enable the acquisition of 2D 1H-13C HETCOR NMR spectra. 2D 1H-13C HETCOR experiments were applied to resolve and partially assign the NMR signals of the form I and form II polymorphs of aspirin in a sample containing both forms. These findings have important implications for ultra-high field NMR experiments, optimization of decoupling schemes and assessment of the fundamental limits on the resolution of solid-state NMR spectra.

Metabolic Profiling of Human In-patient Stools: The Influence of C. difficile Infection and Recurrence

BackgroundInitial C. difficile infection (CDI) and recurrence is dependent on host susceptibility. Hospital patients have a diverse range of other comorbidities and concomitant medications that cannot be duplicated in animal models. We hypothesize that the intersection of the luminal biome and host is represented by the metabolites. Their contribution facilitates CDI clearance or recurrence.MethodsDiscarded stool samples from 4 groups of in-patients (UC IRB 2014–8646) were used. Case controls (CC,n = 20) have diarrhea but are CDI-. Initial CDI+ (I, n = 20) clear infection. Initial CDI+ who become recurrent (IR, n = 18) have samples from initial infection. Known CDI+ Recurrent (KR, n = 10) have samples from second to fourth CDI. Stools were kept at 4°C until aliquoted, weighed and frozen at -80°C. We lyophilized each aliquot and performed metabolome analysis on 50 mg of dry sample. The NMR-Based Metabolomics Core used 1D NOESY and 1D CPMG NMR experiments to generate raw metabolic concentrations. Metabolite IDs were validated with 2D 1H-1H TOCSY and 2D 1H-13C HSQC experiments.Results46 metabolites could be definitively identified and compared. Analysis of raw metabolite mM concentrations in CDI+ stools demonstrated more acetate (median ± median absolute deviation) (CC: 1.63 ± 1.54, I: 5.20 ± 3.21, IR: 4.21 ± 1.46, KR: 2.4 ± 2.15) and butyrate (CC: .1195 ± 0.1185, I: 0.4992 ± 0.3721, IR: 0.4196 ± 0.3832, KR: 0.2279 ± 0.2109). This suggests inability of host’s colon to productively use these SCFA. Levels of stool propionate appear to be linked to the recurrent environment (CC: 0.7620 ± 0.6562, I: 1.5162 ± 0.9189, IR: 0.6928 ± 0.5369, KR: 0.4490 ± 0.3406). Tryptophan catabolism has been linked to the CDI neutrophil response in mice. We detected decreased tryptophan in KR stool as well as lower numbers of circulating neutrophils (KR: 0.007 ± 0.007, CC: 0.0229 ± 0.0199, I: 0.0163 ± 0.0163, IR: 0.0246 ± 0.0201). This suggests altered host luminal metabolism can lead to biosystem changesConclusionCDI in the human biosystem is complex with contribution of host comorbidities and medications. The luminal interface between biome and host is the metabolome. NMR metabolomics of human in-patient stools can provide a more relevant understanding of what conditions are necessary for CDI, its clearance and recurrence. Defeat of this costly infection needs alternative approaches.Disclosures M. B. Yacyshyn, Merck: Grant Investigator, Research grant; B. Yacyshyn, Merck: Grant Investigator and Speaker’s Bureau, Research grant and Speaker honorarium

Nuclear Magnetic Resonance (NMR) Study for the Detection and Quantitation of Cholesterol in HSV529 Therapeutic Vaccine Candidate

This study describes the NMR-based method to determine the limit of quantitation (LOQ) and limit of detection (LOD) of cholesterol, a process-related impurity in the replication-deficient Herpes Simplex Virus (HSV) type 2 candidate vaccine HSV529. Three signature peaks from the 1D 1H NMR of a cholesterol reference spectrum were selected for the identification of cholesterol. The LOQ for a cholesterol working standard was found to be 1μg/mL, and the LOD was found to be 0.1μg/mL. The identity of cholesterol, separated from the formulation of growth supplement by thin layer chromatography (TLC), was confirmed by 1D 1H NMR and 2D 1H-13C HSQC NMR. The three signature peaks of cholesterol were detected only in a six-times concentrated sample of HSV529 candidate vaccine sample and not in the single dose HSV529 vaccine sample under similar experimental conditions. Taken together, the results demonstrated that NMR is a direct method that can successfully identify and quantify cholesterol in viral vaccine samples, such as HSV529, and as well as in the growth supplement used during the upstream stages of HSV529 manufacturing.

Synthesis, characterization, and electrospinning of novel polyaniline–peptide polymers

Aniline–peptide (FLDQV, FLDQVC, Dansyl-FLDQV, Dansyl-FLDQVC, and FLDQV-AMC) mixtures underwent oxidative chemical and electrochemical polymerization in excess of aniline. The products of the chemical polymerization were low molecular weight polymers containing more than 70% peptide. Electrochemically polymerized species polyaniline-FLDQV (PANI-FLDQV) consisted mainly of polyaniline units containing about 10% peptide. The solubility of the latter in 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) was similar to the camphorsulfonic acid (CSA) doped emeraldine base (PANI-CSA) solubility, however the weight composition of the electrospun fibers produced from the two polymers was significantly different. 2D 1H–13C HSQC analyses were employed to analyze the binding between the aniline and peptide moieties. Binding of peptide to polyaniline is reflected by the appearance of extra cross-peaks which display line broadening between the free polyaniline and the free pentapeptide. Peptides may be chemically bonded to the polymer molecules, but they may also act as doping agents to the nitrogen atoms via hydrogen bonding.

NMR-based metabolomics reveals urinary metabolome modifications in female Sprague–Dawley rats by cranberry procyanidins

A 1H NMR global metabolomics approach was used to investigate the urinary metabolome changes in female rats gavaged with partially purified cranberry procyanidins (PPCP) or partially purified apple procyanidins (PPAP). After collecting 24-h baseline urine, 24 female Sprague–Dawley rats were randomly separated into two groups and gavaged with PPCP or PPAP twice using a dose of 250 mg extracts per kilogram body weight. The 24-h urine samples were collected after the gavage. Urine samples were analyzed using 1H NMR. Multivariate analyses showed that the urinary metabolome in rats was modified after administering PPCP or PPAP compared to baseline urine metabolic profiles. 2D 1H-13C HSQC NMR was conducted to assist identification of discriminant metabolites. An increase of hippurate, lactate and succinate and a decrease of citrate and α-ketoglutarate were observed in rat urine after administering PPCP. Urinary levels of d-glucose, d-maltose, 3-(3′-hydroxyphenyl)-3-hydroxypropanoic acid, p-hydroxyphenylacetic acid, formate and phenol increased but citrate, α-ketoglutarate and creatinine decreased in rats after administering PPAP. Furthermore, the NMR analysis showed that the metabolome in the urine of rats administered with PPCP differed from those gavaged with PPAP. Compared to PPAP, PPCP caused an increase of urinary excretion of hippurate but a decrease of 3-(3′-hydroxyphenyl)-3-hydroxypropanoic acid, p-hydroxyphenylacetic acid and phenol. These metabolome changes caused by cranberry procyanidins may help to explain its reported health benefits and identify biomarkers of cranberry procyanidin intake.

Low temperature hydrogenation of pyrolytic lignin over Ru/TiO2: 2D HSQC and 13C NMR study of reactants and products

The low temperature hydrogenation of pyrolytic lignin over Ru/TiO2 was studied and characterized with quantitative 13C and 2D 1H–13C HSQC NMR to determine the changes in carbon functionality.

CO2 activation through silylimido and silylamido zirconium hydrides supported on N-donor chelating SBA15 surface ligands.

Density functional theory calculations and 2D 1H-13C HETCOR solid state NMR spectroscopy prove that CO2 can be used to probe, by its own reactivity, different types of N-donor surface ligands on SBA15-supported Zr(IV) hydrides: [≡(Si-O-)(≡Si-N=)[Zr]H] and [≡(Si-NH-)(≡Si-X-)[Zr]H2] (X=O or NH). Moreover, [≡(Si–O-)(≡Si-N=)[Zr]H] activates CO2 more efficiently than the other complexes and leads to the formation of a carbimato Zr formate.

17β-estradiol (E2) in membranes: Orientation and dynamic properties

Non-genomic membrane effects of estrogens are of great interest because of the diverse biological activities they may elicit. To further our understanding of the molecular features of the interaction between estrogenic hormones and membrane bilayers, we have determined the preferred orientation, location, and dynamic properties of 17β-estradiol (E2) in two different phospholipid membrane environments using 2H-NMR and 2D 1H-13C HSQC in conjunction with molecular dynamics simulations. Unequivocal spectral assignments to specific 2H labels were made possible by synthesizing six selectively deuterated E2 molecules. The data allow us to conclude that the E2 molecule adopts a nearly “horizontal” orientation in the membrane bilayer with its long axis essentially perpendicular to the lipid acyl-chains. All four rings of the E2 molecule are located near the membrane interface, allowing both the E2 3-OH and the 17β-OH groups to engage in hydrogen bonding and electrostatic interactions with polar phospholipid groups. The findings augment our knowledge of the molecular interactions between E2 and membrane bilayer and highlight the asymmetric nature of the dynamic motions of the rigid E2 molecule in a membrane environment.

Changes in Lignin and Polysaccharide Components in 13 Cultivars of Rice Straw following Dilute Acid Pretreatment as Studied by Solution-State 2D 1H-13C NMR.

A renewable raw material, rice straw is pretreated for biorefinery usage. Solution-state two-dimensional (2D) 1H-13 C hetero-nuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy, was used to analyze 13 cultivars of rice straw before and after dilute acid pretreatment, to characterize general changes in the lignin and polysaccharide components. Intensities of most (15 of 16) peaks related to lignin aromatic regions, such as p-coumarate, guaiacyl, syringyl, p-hydroxyphenyl, and cinnamyl alcohol, and methoxyl, increased or remained unchanged after pretreatment. In contrast, intensities of most (11 of 13) peaks related to lignin aliphatic linkages or ferulate decreased. Decreased heterogeneity in the intensities of three peaks related to cellulose components in acid-insoluble residues resulted in similar glucose yield (0.45–0.59 g/g-dry biomass). Starch-derived components showed positive correlations (r = 0.71 to 0.96) with glucose, 5-hydroxymethylfurfural (5-HMF), and formate concentrations in the liquid hydrolysates, and negative correlations (r = –0.95 to –0.97) with xylose concentration and acid-insoluble residue yield. These results showed the fate of lignin and polysaccharide components by pretreatment, suggesting that lignin aromatic regions and cellulose components were retained in the acid insoluble residues and starch-derived components were transformed into glucose, 5-HMF, and formate in the liquid hydrolysate.

Amberinone, a new guaianolide from Amberboa ramosa

The Amberboa is a medicinally important genus present in the family Asteraceae; members of this genus are mainly distributed in Pakistan and India. It has been used in different systems of traditional medicines for different diseases. Amberinone (1), a new sesquiterpene lactone, has been isolated from the ethyl acetate (EtOAc) soluble fraction of Amberboa ramosa together with chrysin (2), quercitine (3), eriodictyol (4) and keamferol (5). This is the first report of these compounds from this species. The structures of the isolated compounds have been elucidated by 1D and 2D 1H 13C NMR spectroscopy.

Identification of metabolites from 2D (1)H-(13)C HSQC NMR using peak correlation plots.

BackgroundIdentification of individual components in complex mixtures is an important and sometimes daunting task in several research areas like metabolomics and natural product studies. NMR spectroscopy is an excellent technique for analysis of mixtures of organic compounds and gives a detailed chemical fingerprint of most individual components above the detection limit. For the identification of individual metabolites in metabolomics, correlation or covariance between peaks in 1H NMR spectra has previously been successfully employed. Similar correlation of 2D 1H-13C Heteronuclear Single Quantum Correlation spectra was recently applied to investigate the structure of heparine. In this paper, we demonstrate how a similar approach can be used to identify metabolites in human biofluids (post-prostatic palpation urine).ResultsFrom 50 1H-13C Heteronuclear Single Quantum Correlation spectra, 23 correlation plots resembling pure metabolites were constructed. The identities of these metabolites were confirmed by comparing the correlation plots with reported NMR data, mostly from the Human Metabolome Database.ConclusionsCorrelation plots prepared by statistically correlating 1H-13C Heteronuclear Single Quantum Correlation spectra from human biofluids provide unambiguous identification of metabolites. The correlation plots highlight cross-peaks belonging to each individual compound, not limited by long-range magnetization transfer as conventional NMR experiments.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-014-0413-z) contains supplementary material, which is available to authorized users.

Study on Dendrobium officinale O-acetyl-glucomannan (Dendronan®): Part II. Fine structures of O-acetylated residues

Main objective of this study was to investigate the detailed structural information about O-acetylated sugar residues in Dendronan®. A water solution (2%, w/w) of Dendronan® was treated with endo-β-mannanase to produce oligosaccharides rich in O-acetylated sugar residues. The oligosaccharides were partly recovered by ethanol precipitation (70%, w/w). The recovered sample (designated Hydrolyzed Dendrobium officinale Polysaccharide, HDOP) had a yield of 24.7% based on the dry weight of Dendronan® and was highly O-acetylated. A D2O solution of HDOP (6%, w/w) generated strong signals in 1H, 13C, 2D 1H–1H COSY, 2D 1H–1H TOCSY, 2D 1H–1H NOESY, 2D 1H–13C HMQC, and 2D 1H–13C HMBC NMR spectra. Results of NMR analyses showed that the majority of O-acetylated mannoses were mono-substituted with acetyl groups at O-2 or O-3 position. There were small amounts of mannose residues with di-O-acetyl substitution at both O-2 and O-3 positions. Minor levels of mannoses with 6-O-acetyl, 2,6-di-O-acetyl, and 3,6-di-O-acetyl substitutions were also identified. Much information about sugar residue sequence was extracted from 2D 1H–13C HMBC and 2D 1H–1H NOESY spectra. 1JC–H coupling constants of major sugar residues were obtained. Evidences for the existence of branches or O-acetylated glucoses in HDOP were not found. The major structure of Dendronan® is shown as follows:▪

New Insights into the Hydrogen Bond Network in Al-MIL-53 and Ga-MIL-53

Metal−organic framework-type Al-, Ga-, and Ga(OH,F)-MIL-53 have been characterized by solid-state NMR and powder X-ray diffraction (PXRD). 1H 2D double-quantum–single-quantum (DQ–SQ) magic angle spinning (MAS) NMR experiments unambiguously evidence two inequivalent water molecules in Al-MIL-53_np_H2O. A careful reinvestigation of the XRD structure of hydrated Al-MIL-53 proves, for the first time, the doubling of the unit cell supporting the presence of two inequivalent water molecules. One type of water molecule is located in the type A channel, interacting with the aromatic protons of framework organic moieties, whereas the other type of water molecule is in the type B channel far away from aromatic protons. Assignment of the corresponding 1H resonances to each water molecule was possible. 1H 1D MAS NMR leads to the same conclusion (i.e., two inequivalent water molecules) for the isostructural gallium-based material thanks to the positioning of the hydrogen atoms by Rietveld refinement. Moreover, when Ga-MIL-53 is prepared with fluorine in the synthesis medium (giving Ga(OH,F)-MIL-53_np_H2O), the situation is different. 2D 1H–13C heteronuclear correlation MAS NMR, high-field 1H DQ–SQ, and radio-frequency-driven recoupling MAS NMR indicate that only one type of water molecule is found in this material, in agreement with the structure.

Direct nuclear magnetic resonance identification and quantification of geometric isomers of conjugated linoleic acid in milk lipid fraction without derivatization steps: Overcoming sensitivity and resolution barriers

We report the first successful direct and unequivocal identification and quantification of four minor geometric (9-cis, 11-trans) 18:2, (9-trans, 11-cis) 18:2, (9-cis, 11-cis) 18:2 and (9-trans, 11-trans) 18:2 conjugated linoleic acid (CLA) isomers in lipid fractions of lyophilized milk samples with the combined use of 1D 1H-NMR, 2D 1H1H TOCSY and 2D 1H13C HSQC NMR. The significant sensitivity barrier has been successfully overcome under selective suppression of the major resonances, with over 104 greater equilibrium magnetization of the (CH2)n1H spins compared to that of the 1H spins of the conjugated bonds of the CLA isomers. The resolution barrier has been significantly increased using reduced 13C spectral width in the 2D 1H13C HSQC experiment. The assignment was confirmed with spiking experiments with CLA standard compounds and the method does not require any derivatization steps for the lipid fraction. The proposed method is selective, sensitive and compares favorably with the GS-MS method of analysis.

Platinum(IV) complexes with N-alkylphenothiazines: synthesis, characterization, and antibacterial activity

Two new platinum(IV) complexes (1, trifluoperazinehydrochloride-aquapentachloridoplatinate(IV) and 2, chlorpromazine-chlorpromazinehydrochloridepentachloridoplatinate(IV)) were synthesized in the reaction of K2[PtCl6] with trifluoperazine dihydrochloride (TF·2HCl) or chlorpromazine hydrochloride (CP·HCl). The complexes were characterized by elemental analysis, molar conductivity measurement, and spectral (IR, 1H, 13C, 2D 1H–13C heteronuclear correlation spectra, 195Pt NMR, and MS) methods. Outer-coordination sphere was proposed for 1; while in 2, the ligand was coordinated to the metal. The complexes exhibit antibacterial effect on strains of Bacillus subtilis, Bacillus cereus, Bacillus pumilus, and methicillin-resistant Staphylococci as Gram-positive bacteria and an Escherichia coli as Gram-negative bacteria, as well as the reference strains.

Strong Intermolecular Ring Current Influence on 1H Chemical Shifts in Two Crystalline Forms of Naproxen: a Combined Solid-State NMR and DFT Study

The anhydrous crystalline forms of Naproxen [(S)-(+)-2-(6-methoxy-2-naphthyl)propionic acid], (NAPRO-A) and its sodium salt (NAPRO-S), widely used anti-inflammatory drugs, have been investigated by means of 1D and 2D MAS NMR and density functional theory (DFT) based calculations. From calculations, 1D 13C CP MAS and 1H CRAMPS and 2D 1H–13C MAS-J-HMQC, refocused INEPT, FSLG-HETCOR, and 1H–1H DQ-CRAMPS solid-state NMR experiments, 1H and 13C resonances have been fully assigned for NAPRO-A and -S. In the case of NAPRO-S, all of the nuclei belonging to the two inequivalent molecules of the asymmetric cell gave rise to distinct signals, which could be completely assigned. Interesting intermolecular ring current effects on 1H chemical shifts have been experimentally observed for the two samples, even if with significant differences between the two cases. The measured and calculated proton chemical shift values showed a very good agreement for both NAPRO-A and -S, allowing us to correlate the different ring current effects with the crystal structures. The comparison between the proton chemical shifts calculated in the crystal structures and in vacuo allowed us to confirm the mainly intermolecular character of the ring current effects and to quantify them.

“Perfect Echo” HMQC: Sensitivity and resolution enhancement by broadband homonuclear decoupling

Homonuclear 1H–1H J-modulation leads to J-multiplets in F1 dimension of 2D 1H–13C HMQC spectra. This hampers unambiguous signal assignment for overcrowded 13C spectra. Broadband homonuclear decoupling has been achieved in the indirect t1 evolution period by incorporating blocks of perfect echo. This method of perfect echo HMQC demonstrates better resolution and sensitivity than conventional HMQC spectra. The results on Cyclosporine demonstrate that the method is very efficient for refocusing geminal couplings in weakly coupled –13CH2 groups. Partial refocusing of vicinal couplings is also observed for –13CH and –13CH3 groups. Interpretation of the result based on product operator formalism is also given. Comparison of pe-HMQC, HMQC and HSQC reveals that the F1 linewidth of pe-HMQC is much narrower than HMQC and very close to that of HSQC for CH2 groups.

Preparation and characterization of cycloolefin polymer based on dicyclopentadiene (DCPD) and dimethanooctahydronaphthalene (DMON)

Ring-opening metathesis polymerization (ROMP) and subsequent hydrogenation of the mixture of dicyclopentadiene (DCPD) and 1,4,5,8-dimethano-1,2,3,4,4a,5,8,8a-octahydronaphthalene (DMON) were conducted. ROMP was successfully performed at room temperature using WCl6/iBu3Al/2-BuOH (1/0.7/1.2) catalyst system when the DCPD/DMON molar ratio was 1. Depending on the DCPD/DMON molar ratio, the catalyst component ratio for the polymerization changed. The double bond in the prepared ROMP polymer could be completely hydrogenated using Ni(acac)2/iBu3Al at molar ratio of the Ni/double bond in polymer=0.2mol% and Al/Ni=4 when the reaction was conducted at 80°C for 3h. After hydrogenation, the glass transition temperature (Tg) of all ROMP polymers was decreased by 40°C. Depending on the DCPD and DMON molar ratio, the Tg of the ROMP polymer and hydrogenated ROMP polymer changed. The hydrogenated DCPD–DMON ROMP copolymer was characterized by using 2D 1H–1H COSY and 2D 1H–13C HSQC NMR spectroscopy. Thermal stability and light transmittance for the ROMP polymer and the hydrogenated ROMP polymer were compared by using TGA and UV.

Abstract 1886: Metabolic alterations associated with pancreatic cancer-induced cachexia.

Abstract Cachexia, a metabolic syndrome, leads to loss of muscle weight and fat tissues. Cancer-induced cachexia accounts for nearly 20% of all cancer-related deaths and is most prevalent in patients with gastric, pancreatic, colon and lung cancer. Although association of cachexia with various types of cancers has been known for a long time, the molecular mechanism of cancer-induced cachexia is still not well understood. Better understanding of the cancer-related cachexia syndrome may provide novel therapeutic targets and improved health care for cancer patients. By culturing differentiated C2C12 and 3T3-L1 cells in the presence of pancreatic adenocarcinoma cell or immortalized epithelial cell supernatants, we established cell culture-based cachexia model systems to investigate the depletion of muscle mass and fatty acid storage. Progressive physiological and biochemical alterations during muscle fibre breakdown and adipocyte fat depletion were assayed by utilizing western blotting, real-time PCR and cell imaging. We then investigated metabolomic alterations in C2C12 and 3T3-L1 cell lines in response to the cachectic factors secreted by the tumor cells. We performed differential metabolomic analyses on tumor cell supernatant-treated or control treated differentiated C2C12 muscle fibers and 3T3-L1-derived adipocytes by utilizing 2D 1H-13C HSQC NMR technique. Alterations in metabolic pathways were also investigated by evaluating the expression of metabolic genes in the corresponding pathways by performing quantitative real-time PCR. To obtain further insights into the metabolic alterations that trigger cachexia, we extended our studies to in vivo mouse orthotopic xenograft models and investigated alterations in metabolic gene expression and metabolite flux in muscle and fat tissues of tumor-bearing mice. Our results identify crucial nodes in metabolite flux that can be targeted to regulate cell growth, proliferation, and turnover in muscle and fat tissues during cancer-induced cachexia. Citation Format: Surendra K. Shukla, Pankaj K. Singh. Metabolic alterations associated with pancreatic cancer-induced cachexia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1886. doi:10.1158/1538-7445.AM2013-1886