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Metabolomics (2013) 9:101118 DOI 10.

1007/s11306-012-0431-7

ORIGINAL ARTICLE

Magic angle spinning NMR spectroscopic metabolic proling of gall bladder tissues for differentiating malignant from benign disease
Santosh Kumar Bharti Anu Behari Vinay Kumar Kapoor Niraj Kumari Narendra Krishnani Raja Roy

Received: 13 April 2012 / Accepted: 2 May 2012 / Published online: 26 May 2012 Springer Science+Business Media, LLC 2012

Abstract Gall bladder tissue specimens obtained from 112 patients were examined by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. Fifty one metabolites were identied by combination of one and twodimensional NMR spectra. To our knowledge, this is the rst report on metabolic proling of gall bladder tissues using HR-MAS NMR spectroscopy. Metabolic proles were evaluated for differentiation between benign Chronic Cholecystitis (CC, n = 66) and xantho-granulomatous cholecystitis (XGC, n = 21) and malignant gall bladder cancer (GBC, n = 25). Increase in choline containing compounds, amino acids, taurine, nucleotides and lactate as common metabolites were observed in malignant tissues whereas lipid content was found low as compared to benign tissues. Principal component analysis obtained from the NMR data showed clear distinction between CC and GBC tissue specimens; however, 27 % of XGC tissues were classied with GBC. The partial least square discriminant analysis (PLS-DA)

multivariate analysis between benign (CC, XGC) and malignant (GBC) on the training data set (CC; n = 51, XGC; n = 15, GBC; n = 19 tissues specimens) provided 100 % sensitivity and 94.12 % specicity. This PLS-DA model when executed on the spectra of unknown tissue specimens (CC; n = 15, XGC; n = 6, GBC; n = 6) classied them into the three histological categories with more than 95 % of diagnostic accuracy. Non-invasive in vivo MRS technique may be used in future to differentiate between benign (CC and XGC) and malignant (GBC) gall bladder diseases. Keywords HR-MAS NMR spectroscopy Gall bladder cancer (GBC) Xantho-granulomatous cholecystitis (XGC) Chronic cholecystitis (CC) Metabolic proling Metabolomics Abbreviations BCA Branch chain amino acids CC Chronic cholecystitis CPMG Carr-purcell-meiboom-gill DQF-COSY Double quantum ltered-correlation spectroscopy GBC Gall bladder cancer HR-MAS High resolution-magic angle spinning PLS-DA Partial least square regression discriminant analysis PCA Principal component analysis XGC Xantho-granulomatous cholecystitis

Electronic supplementary material The online version of this article (doi:10.1007/s11306-012-0431-7) contains supplementary material, which is available to authorized users.
S. K. Bharti R. Roy (&) Centre of Biomedical Magnetic Resonance, Sanjay Gandhi Postgraduate Institute of Medical Sciences Campus, Raibarely Road, Lucknow 226014, Uttar Pradesh, India e-mail: roy@cbmr.res.in A. Behari V. K. Kapoor (&) Department of Surgical Gastroenterology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raibarely Road, Lucknow 226014, Uttar Pradesh, India e-mail: vkkapoor.india@gmail.com N. Kumari N. Krishnani Department of Pathology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

1 Introduction Gall bladder cancer (GBC) represents the fth most common malignancy of the gastro-intestinal tract and the commonest malignancy of the biliary tract worldwide

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(Misra and Guleria 2006). It has an unusual geographic distribution having more frequency in Chile, Bolivia, Israel and northern India than in United States and Europe (Gupta and Shukla 2005; Orth and Beger 2000). According to the Indian Council for Medical Research (ICMR) reports, the incidence rate of GBC is very high in northern India with the highest in world and therefore GBC could be named an Indian disease (Kapoor 2007). GBC is 23 times more common in women than men (Yalcin 2004); the incidence of GBC in India is 10 times more frequent in women that is 10.1 per 100,000 for females and 1.01 per 100,000 for males (ICMR 1996). Prognosis of GBC is very poor and about 40 % of the patients have survival rate of ve years in case of early diagnosis and one year in case of advanced stages (Lazcano-Ponce et al. 2001). Chronic cholecystitis (CC) is inammation of gall bladder (GB) which is associated with gall stones in more than 90 % of the cases (Schirmer et al. 2005). It occurs when gallstone or solid sludge impact in the cystic duct and inammation develops behind the obstruction (Elwood 2008). Xanthogranulomatous cholecystitis (XGC), an uncommon variants of chronic cholecystitis (Yang et al. 2007); (Chang et al. 2010; Roberts and Parsons 1987) is characterized by thickening of gall bladder wall, focal or destructive inammation with accumulation of lipid laden macrophages, brous tissue, with acute and chronic inammatory cells (Jessurun and Albores-Saavendra 1996). Clinically, it is very difcult to distinguish XGC from any other inammatory gall bladder disease such as CC, acute cholecystitis and especially GBC. The XGC mimics GBC which makes it difcult to differentiate by imaging techniques like ultrasonography (US), computed tomography (CT) and magnetic resonance imaging (MRI) (Chang et al. 2010). The nal diagnosis is obtained only through histopathological examination. The exact etiology of GBC is unknown, but several risk factors have been identied including age, gender, cholelithiasis, chronic inammation of gall bladder, increased stone size, family history, choledochal cyst, composition of bile acids, infection, exposure to carcinogens etc. (Gupta and Shukla 2005; Tazuma and Kajiyama 2001; Pandey et al. 1995). The most accepted model for development of GBC includes initial chronic inammation leading to metaplasia, dysplasia, carcinoma in situ and nally to invasive cancer (Roa et al. 2009; Roa et al. 2006). Chronic inammation may arise due to gall stone obstruction, bacterial infection, metabolic disturbances etc. (Roa et al. 1996). Discrimination between the benign (CC and XGC) and malignant (GBC) has an important role in management of patients care. Metabolomics allows the qualitative and quantitative measurements of all metabolites present in cells, biouids, pathological uids, tissues, tissue extracts etc. (Hollywood et al. 2006; Lindon et al. 2004). Common analytical

techniques used for metabolomics studies are HPLC, MS, GC, GCMS and NMR spectroscopy. Among them high resolution NMR spectroscopy is widely used for investigating the composition of body uids, tissues extracts, pathological uids etc. as a wide range of metabolites can be detected simultaneously without separation of individual components (Lindon et al. 2000). NMR based metabolic proling followed by pattern recognition statistical techniques provides a comprehensive metabolic information of various components in biouids, reecting levels of endogenous metabolites/biomarkers involved in key cellular pathways, which indicate physiological and pathophysiological status, and also further used in diagnosis (Lindon and Nicholson 2008). High-resolution magic angle spinning (HR-MAS) NMR spectroscopy is a further advancement of this technique and it provides fast, easy and direct analysis of intact tissues cells, foods, paste, soils, fruit, plant, tissues (Beckonert et al. 2010; Bharti et al. 2011) etc. It also offers qualitative and quantitative biochemical information on small intact tissue samples by generating metabolic prole. The HR-MAS NMR analysis of tissue specimens allows the simultaneous detection of both lipids and small metabolites with a resolution comparable to that of liquid state NMR. The HR-MAS NMR spectroscopy is nondestructive unlike extraction procedures and tissue specimens can be further used for routine molecular biology experiments (Stenman et al. 2010). Extraction procedure requires large quantity of the samples and increases the chance of loss of some low concentrated metabolites or reduction in their intensity which leads to the misinterpretation of data (Duportet et al. 2011; Cheng et al. 1998). In recent years, HR-MAS NMR spectroscopy has been successfully applied to characterize the metabolic composition of control and pathological tissues from brain (Wright et al. 2010), pancreas (Misra et al. 2008), lung (Rocha et al. 2009), breast (Gribbestad et al. 1994), colorectal cancer (Chan et al. 2009) etc. Therefore in the present study, 1H HR-MAS NMR based metabolomic approach has been applied with an aim to carry out metabolic proling of gall bladder tissues followed by chemometric and quantitative analysis, for discrimination of benign and malignant tissues types.

2 Materials and methods 2.1 Subjects and study protocol Gall bladder tissue specimens (n = 112, CC = 66, XGC = 21, GBC = 25) were collected from patients undergoing laparoscopic cholecystectomy/open cholecystectomy at the Department of Surgical Gastroenterology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, a

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tertiary care super specialty hospital in northern India. Gall bladder tissues specimens, conrmed to have CC, XGC and GBC on histo-pathology were included in this study. Tissue samples from male and female patients with age more than 18 and \70 years were taken after surgical removal. The suspected region chosen by surgeons were visibly resected and then subjected for histopathology and NMR analysis. A Part of the same tissues specimen was sent to the department of pathology for routine histopathological testing and other part for NMR analysis. All samples were snap frozen in liquid nitrogen within 15 min after surgery and stored at -80 C until NMR spectra were recorded. The study has been approved by SGPGIMS institute ethics committee and consent from each patient was obtained prior to investigations. 2.2 Sample preparation and acquisition Tissue specimen stored at -80 C were thawed at room temperature and then washed with saline deuterated water in order to remove blood from tissues specimens. Dissected and weighed pieces of gall bladder were inserted in the ZrO2 rotor of 50 ll. A volume of 20 ll of D2O was lled in the rotor with tissue sample for locking the spectrometer frequency. The sample-rotor-setup was then transferred in the HR-MAS probe. A sample weight of 32 1.5 mg of wet tissue was used for analysis. After NMR analysis all the tissue specimens were xed in formalin in order to observe the high spinning effect on the tissue integrity by histopathological examination. 2.3 NMR experimental conditions H NMR spectra were recorded on Bruker Biospin AvanceIII 800 MHz NMR (Bruker GmBH, Germany) spectrometer operating at proton frequency of 800.21 using 4 mm HRMAS 1H/13C/31P triple resonance probehead equipped with magic angle gradient accessories. A zirconium oxide rotor of 4 mm diameter was used for the spectral recording with a spinning speed of 8000 2 Hz. Twenty microliter of deuterium oxide containing (Sigma-Aldrich, St. Louis, MO, USA) was used for internal lock. Sample temperature was regulated using Bruker BCU-05 unit at 280 0.5 K during the acquisition of spectra to reduce the metabolic changes during spectral acquisition (Beckonert et al. 2010). The calibration of the temperature was performed using methanol during setup of the HR-MAS probe. HR-MAS NMR spectra were recorded within few days and no further temperature calibration was performed during the sample analysis. 2.3.1 One dimensional NMR analysis The 1H HR-MAS spectra with water suppression were acquired using one-dimensional single pulse and Carr1

Purcell-Meiboom-Gill (CPMG) pulse sequence with the following experimental parameters: spectral width of 12,820.5 Hz,, time domain data points of 64 K, effective 90 ip angle, 9.0 ls, relaxation delay 4.0 s acquisition time of 2.55 s, 64 number of scan with 4 dummy scan, a constant receiver gain of 50.8 with a total recording time of 9 min. CPMG pulse sequence with water suppression [PRESET-90-(d-180-d)n-Aq] was performed to remove short T2 components arising due to the presence of proteins as well as to obtain a good baseline for multivariate analysis. Echo time of 40 ms (2d 9 n, n = 200, d = 100 ls) was used in CPMG pulse sequence. All spectra were processed using line broadening for exponential window function of 0.3 Hz prior to Fourier transformation. The 1H HR-MAS spectra of gall bladder tissues were manually phased and automatically baseline corrected using TOPSPIN 2.1 (Bruker Analytik, Rheinstetten, Germany). The 1 H NMR spectra were referenced to the methyl resonance of alanine at 1.48 ppm. The total analysis time (including sample preparation, optimization of NMR parameters and data acquisition) of 1H HR-MAS NMR spectroscopy for each sample was approximately 20 min. 2.3.2 Two dimensional NMR analysis To conrm the assignments, two-dimensional homo nuclear correlation spectroscopy (1H-1H COSY) and 1 H-13C hetero nuclear single quantum correlation spectroscopy (HSQC) experiments were performed using Brukers standard pulse program library. The parameters used for COSY were as follows: 2 K data points were collected in the t2 domain over spectral width of 12820 Hz, 512 t1 increments were collected with 64 transients, relaxation delay of 1.5 s, acquisition time of 95 ms, and pre-saturation of water resonance was carried out during the relaxation delay. The resulting data were zero-lled to 1 K and were weighted with sine bell window functions in both the dimensions prior to Fourier transformation. The parameters used for 1H-13C HSQC were: 2 K data points were collected in t2 dimension over spectral width of 12,820 Hz, 256 t1 increments were collected with 32 transients, relaxation delay of 2.0 s, acquisition time of 80 ms and a 90 pulse of 9.0 ls. The phase sensitive data were obtained by the antiecho-Time proportional phase increments (Antiecho-TPPI) method. The resulting data were zerolled to 512 data points and were weighted with 90 shifted squared sine bell window functions in both the dimensions prior to Fourier transformation. 2.4 Statistical analysis Multivariate principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) was

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performed on the HR-MAS NMR data. The external validation of the data was performed by dividing about 75 % of the patients as training set and the remaining 25 % as test set. In the present study, 85 samples obtained from 85 patients were randomly selected from Fisher and Yates table (Fisher and Yates 1957) as training set for PLS-DA model generation and the rest 27 samples from 27 patients were predicted on the basis of that model. The training set comprised of 85 tissue specimens with 66 tissues (CC; n = 51, XGC; n = 15) having benign histology while rest 19 (GBC; n = 19) were malignant in nature. The 27 tissues, comprising the test set, were not included during construction of the model and treated as blinded samples till the prediction of their respective pathological status and then compared with their corresponding histopathology reports. Before subjecting the CPMG spectra for multivariate analysis, these were reduced to discrete chemical shift regions (between 0.5 and 9.0 ppm) by digitization to produce a series of sequentially integrated regions of 0.01 ppm width, using Bruker AMIX software (Version 3.8.7, Bruker Biospin, Germany). Simple rectangular bucketing procedure was chosen to integrate the peak area. The data obtained was mean centered and normalized by dividing each integral area of the segment by total area of the spectrum in order to compensate for the differences in overall metabolite concentration between individual samples. The resulting data matrices having normalized integral values were exported into Microsoft Ofce Excel 2007 (Microsoft Corporation, USA). This was further imported to The Unscrambler X Software package (Version 10.0.1, Camo USA) for multivariate Principal Component Analysis (PCA) and Partial Least Square Discriminant Analysis (PLS-DA) analysis. In PCA and PLS-DA, a full cross validation using leave one out were applied in order to avoid the overtting of the mathematical model. To validate the robustness of the PLS-DA model, unknown data set obtained from 27 patients was subsequently analysed. Univariate analysis of semi-quantitative data was performed using MannWhitney U test (SPSS 15.0). 2.5 Semi-quantitative analysis Absolute integral area of resolved metabolites were quantied with respect to QUANTAS (QUANTication by Articial Signal) (Bharti and Roy 2012). QUANTAS is an articial signal generated by NMRSIM (or any NMR simulation software) with a xed line width and intensity was added to real HR-MAS NMR spectrum. The main condition for the QUANTAS is all spectra should be recorded at same acquisition parameters using same receiver gain setting. Integration of some of the metabolites which are not overlapped, were performed for

quantication of absolute intensity and metabolites integral area/intensity were normalized with QUANTAS which provides information about variation in the quantitative values of metabolites in different groups. Mean of integral area with standard deviation was calculated for comparing the CC, XGC and GBC individually.

3 Results Hundred twelve patients were included in the study. Chronic Cholecystitis was more frequent as compared to the XGC and GBC. The routine histopathologies of the surgically resected GBC tissues were found to be adenocarcinoma in nature. The number of females cases were comparatively more in each groups whereas as there was no signicant difference in the mean age of male and female. The detailed distribution of patients in each group, mean age, range and gender are reported in Supplementary Table S-1. 3.1 Metabolic prole of gall bladder tissues A typical proton HR-MAS NMR spectrum of GBC tissues along with assignments is shown in Fig. 1. Using HR-MAS NMR spectroscopy, 51 endogenous metabolites were assigned that includes lipids, amino acids, organic acids, choline containing compound, creatine, sugars, etc. The detailed list of metabolites and assignments is presented in Table 1. Characterization of the metabolites was carried out on the basis of chemical shift, coupling constant, and splitting pattern of metabolites as reported in literature (Gribbestad et al. 1994; Sitter et al. 2006; MartinezGranados et al. 2011; Rocha et al. 2009), two dimensional NMR spectra (Supplementary Fig. 1 and 2), comparison with standard NMR spectra of metabolites reported at Biological Magnetic Resonance Bank (BMRB, www.bmrb. wisc.edu) and Human Metabolome Data Base (HMDB, www.hmdb.ca) (Markley et al. 2007; Wishart et al. 2009). Metabolic prole of malignant (GBC) differed from benign conditions (CC and XGC). The stack plot of one dimensional proton NMR spectra of CC, XGC and GBC with assignment of resonances is shown in Fig. 2. In order to examine the effect of high spinning, few tissues samples were subjected for further histopathological examination after HR-MAS analysis. The tissue has not lost its integrity but the lining epithelium is denuded. The denudation (shedding off) of lining epithelium occurs when there is poor xation or due to high spinning speed. Comparison of routine histopathological examination with histopathology after HR-MAS analysis obtained from the same patient is shown in Fig. 3, which clearly demonstrates similar histopathological ndings.

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Phenylalanine/Tryptophan

Adenine

Tyrosine

Tyrosine

Inosine/Adenosine

Inosine/Adenosine

Tryptophan/Uracil

Inosine/Adenosine

Uracil

8.5

8.0
Taurine Choline Containing Compounds

7.5

7.0

6.5

6.0

5.5

Fatty Acid CH=CH

Histidine Tryptophan

Histidine

Formate

Fumaric Acid

Uridine

Glucose

ppm

Lysine/Creatine

Alanine

Alanine

Lactate

Creatine

Glycine

Methionine Glutamic Acid Proline

Threonine

Myinositol

Lysine

Lysine

Lactate

Glutamic Acid

-Hydroxybutyrate

Glucose

Aspartic Acid

Uridine

Methionine

Valine/Leucine/Isoleucine

Taurine

Asparagine

Ascorbic Acid

Glutamine

Cholesterol

Tyrosine

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

ppm

Fig. 1 A typical 800 MHz 1H HR-MAS NMR spectrum of gall bladder carcinoma (malignant) tissue recorded using CPMG pulse sequence showing assignments of metabolites. Expansions of NMR spectrum from a 0.54.7 and b 5.09.0 ppm

3.2 PCA of CC, XGC and GBC NMR spectra Multivariate principal component analysis (PCA) was performed on the one-dimensional CPMG HR-MAS NMR spectra of CC, XGC and GBC as it provides better baseline. A four principal component model explained [95 % of the variance, with the rst two components explaining 89 % of the total variance. A clear clustering separation between CC and GBC groups in the PCA of spectra demonstrated signicant metabolic variations in CC and GBC groups (Fig. 4A) whereas 27 % of the XGC samples were found to be overlapped with GBC groups and rest of them were

classied with CC. The detailed examination of PC1 (principal components), PC2 and PC3 loadings showed that the cluster separation arising mainly due to TAG signals (Fatty Acid; FA); terminal methyl, (CH3, 0.90 ppm), saturated methylene ((CH2)n, 1.30 ppm), methylene attached with carbonyl methylene (CH2CH2CO, 1.59 ppm), monoallylic methylene (CH2CH=CH, 2.05 ppm), carbonyl methylene (CH2CO, 2.27 ppm), di-allylic methylene (CH=CHCH2CH=CH, 2.78 ppm), TAG-glycerol backbone (TAG-Glycerol, 4.304.13 ppm), branch chain amino acids (BCA; isoleucine, leucine and valine, 0.951.05 ppm), beta-hydroxybutyrate (1.20 ppm), lactate (1.33, 4.12 ppm),

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106 Table 1 Chemical shift assignments of metabolites observed in the HR-MAS spectrum of gallbladder tissues using one dimensional (1D) chemical shift reported in literature (LIT), two dimensional COSY, S. No. 1 2 3 4 5 Name of metabolites Acetate Adenine Alanine Beta-alanine Arginine Chem. shift 1.92 (s) 8.21 (s) 8.23 (s) 1.48 (d) 3.78 (q) 2.56 3.18 1.68 (m) 1.90 (m) 3.25 3.77 6 Ascorbic acid 4.07 3.73 4.55 (s) 7 Asparagine 2.87 (dd) 2.95 (dd) 4.01 (dd) 8 Aspartic acid 2.69 (dd) 2.82 (dd) 3.90 (dd) 9 10 Cholesterol Choline 0.72 (s) 3.21 (s) 3.53 4.07 11 12 13 14 15 Citric acid Creatine Ethanol Ethanolamine Fatty acids (TAG) 2.53 (d) 2.67 (d) 3.03 3.94 1.18 (t) 3.62 3.15 0.90/0.96 1.3 1.59 2.04/2.07 2.26 2.81 4.13 4.31 5.33 16 17 Formic acid Fumaric acid 8.45 (s) 6.52 (s)

S. K. Bharti et al. HSQC and comparing standard NMR spectrum (STD) of individual metabolites taken from Biological Magnetic Resonance Bank (BMRB) Resonances CH3 4H 8H b-CH3 a-CH b-CH2 a-CH2 c-CH2 b-CH2 d-CH2 a-CH C5H CH2 C4H-ring b-CH b0 -CH a-CH b-CH b0 -CH a-CH C18H N(CH3)3 N-CH2 O-CH2 CH2 CH2 N-CH3 N-CH2 CH3 CH2 N-CH2 CH3 (CH2)n CH2CH2CO CH=CHCH2 CH2CO CH=CHCH2CH=CH CH2CHOHCH2 CH2CHOHCH2 CH=CH/CH2CHOHCH2 CH CH 1D, STD 1D, STD 1D, COSY, STD, HSQC 1D, COSY, LIT 1D, COSY, STD 1D, HSQC 1D, STD 1D, STD 1D, COSY, HSQC 1D, COSY, HSQC 1D, COSY, HSQC 1D, STD 1D, COSY, HSQC 1D, COSY, HSQC 1D, COSY, HSQC Methods 1D, HSQC 1D, STD

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Metabolic prole of gall bladder tissues by HR-MAS NMR Table 1 continued S. No. 18 Name of metabolites a-Glucose Chem. shift 3.41 3.54 3.71 3.83 3.85 5.24 (d) 19 b-Glucose 3.24 (dd) 3.41 3.47 3.49 (t) 3.74 3.91 20 Glutamic acid 4.65 (d) 2.09 2.35 (m) 3.77 21 Glutamine 2.13 (m) 2.44 (m) 3.77 22 Glutathione reduced 2.17 (m) 2.56 (m) 2.95 (m) 3.77 (m) 3.78 (m) 4.58 (m) 23 Glycerol 3.56 3.65 24 Glycerol in lipids 3.78 4.09 4.30 5.24 25 26 Glycerolphosphoethanolamine Glycerophosphocholine (GPC) 3.29 4.12 3.24 (s) 3.7 4.32 27 28 Glycine Histidine 3.56 (s) 3.15 (dd) 3.24 (dd) 4.00 (dd) 7.13 (s) 7.98 (s) 29 b-Hydroxybutyrate 1.20 (d) 2.31 (dd) 2.43 (dd) 4.13 30 Hypotaurine 2.65 (t) 3.36 (t) Resonances C4H C2H C3H C6H C5H C1H C2H C4H C5H C3H C6H C60 H C1H b-CH2 c-CH2 a-CH b-CH2 c-CH2 a-CH b-CH2 Glu a-CH2 Cys a-CH2 Gly a-CH2 Glu c-CH2-Glu b-CH Cys CH2 CH20 CH C1H2/C3H2 C1H2/C3H2 C2H N-CH2 O-CH2 N(CH3)3 N-CH2 P-CH2 a-CH2 b-CH b0 -CH a-CH C4H-ring C2H-ring CH3 b-CH b0 -CH a-CH S-CH2 N-CH2 1D, STD, COSY 1D, STD, COSY 1D, STD, HSQC 1D, HSQC, STD 1D, COSY, STD 1D, STD, HSQC 1D, HSQC, STD 1D, COSY, STD 1D, COSY, HSQC 1D, COSY, HSQC 1D, COSY, HSQC Methods 1D, COSY, HSQC

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108 Table 1 continued S. No. 31 Name of metabolites Inosine/adenosine Chem. shift 3.86 4.28 4.44 4.78 (t) 6.10 (d) 8.23 (s) 8.36 (s) 32 Isoleucine 0.94 (t) 1.01 (d) 1.26 (m) 1.47 (m) 1.98 (m) 33 34 35 Isobutyrate Lactate Leucine 3.68 (d) 1.14 (d) 3.88 1.33 (d) 4.12 (q) 0.96 (d) 0.97 (d) 1.71 (m) 3.75 36 Lysine 1.47 (m) 1.72 (m) 1.9 (m) 3.02 3.74 37 Methionine 2.13 (s) 2.16 (s) 2.64 (t) 3.85 38 Myo-inositol 3.28 (t) 3.54 (d) 3.62 (t) 4.06 (t) 39 Phenylalanine 3.12 3.28 3.98 7.32 (d) 7.37 (m) 7.41 (m) 40 Phosphocholine 3.22 3.62 4.18 Resonances C50 500 H ribose C40 H ribose C30 H ribose C20 H ribose C10 H ribose C2H ribose C8H ribose d-CH3 c-CH3 c-CH c0 -CH b-CH a-CH CH3 CH b-CH3 a-CH d-CH3 d0 -CH3 c-CH/b-CH2 a-CH c-CH2 b-CH2 d-CH2 N-CH2 a-CH S-CH3 b-CH2 c-CH2 a-CH C2H-ring C1, 3H-ring C5H-ring C4, 6H-ring b-CH b -CH a-CH C2H, C6H-ring C4H-ring C3H, C5H-ring N(CH3)3 N-CH2 O-CH2
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Methods 1D, STD

1D, COSY, STD, HSQC

1D, STD, COSY 1D, STD, COSY, HSQC 1D, STD, COSY, HSQC

1D, STD, COSY, HSQC

1D, COSY, STD, HSQC

1D, COSY, STD, HSQC

1D, STD, COSY, HSQC

1D, STD, HSQC

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Metabolic prole of gall bladder tissues by HR-MAS NMR Table 1 continued S. No. 41 Name of metabolites Proline Chem. shift 2.01 (m) 2.08 (m) 2.35 (m) 3.35 3.42 4.13 42 43 44 45 46 Serine Scyllo-inositol Succinic acid Taurine Threonine 3.85 3.97 3.35 (s) 2.41 (s) 3.25 (t) 3.41 (t) 1.34 (d) 3.6 (d) 4.25 (m) 47 Tryptophan 3.29 (dd) 3.47 (dd) 4.04 (dd) 7.19 (t) 7.26 (t) 7.30 (s) 7.53 (d) 7.72 (d) 48 Tyrosine 3.06 (dd) 3.19 (dd) 3.95 (dd) 6.89 (d) 49 50 Uracil Uridine 7.18 (d) 5.8 (s) 7.54 (d) 4.13 4.23 (t) 4.35 (t) 5.89 (d)/5.92 (d) 7.89 (d) 51 Valine 0.99 (d) 1.04 (d) 3.62 (d) 2.28 Resonances c-CH2 b-CH b0 -CH d-CH d0 -CH a-CH a-CH b-CH2 CH a,b-CH2 S-CH2 N-CH2 c-CH3 a-CH b-CH b-CH b0 -CH a-CH C5H-ring C6H-ring C2H-ring C4H-ring C7H-ring b-CH b -CH a-CH C3H, C5H-ring C2H, C6H-ring C5H-ring C6H-ring C40 H-ribose C30 H-ribose C20 H-ribose C10 H-ribose/C5H-ring C6H-ring c-CH3 c0 -CH3 b-CH a-CH 1D, COSY, STD
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1D, COSY, HSQC 1D, STD, HSQC 1D, STD, HSQC 1D, COSY, STD, HSQC 1D, COSY, STD, HSQC

1D, COSY, STD, HSQC

1D, COSY, STD, HSQC

1D, COSY, STD, HSQC

1D, COSY, STD, HSQC

alanine (1.48 ppm), creatine (3.03 ppm), choline containing compounds (3.203.24 ppm), taurine (3.25, 3.41 ppm), glycine (3.56 ppm) and glucose (4.65 ppm). TAG components were signicantly very high in CC whereas very low in GBC tissues. Whereas negative loadings in PC1 are due to isoleucine, leucine, valine, lactate, alanine, lysine, creatine, choline containing compounds, taurine, glycine and glucose which demonstrate that these metabolites were high in GBC

samples (Fig. 4B). Few of the XGC spectra were found to be overlapped with GBC and and the rest with CC groups. The detailed individual analysis of each HR-MAS spectrum of XGC samples conrms that the samples overlapped with CC groups have higher content of TAG resonances and resembled to CC tissues HR-MAS spectra. However, XGC samples overlapped with GBC had lower TAG contents, resembling to HR-MAS spectra of GBC.

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Unknown

F
Unknown

Phenylalanine Histidine

Inosine/Adenosine

Nucleotides

Tyrosine

Farmate

Tyrosine

Histidine Tryptophan

Fumaric Acid

Uracil

beta-hydroxybutyrate, lactate, alanine, lysine, creatine, choline compounds, taurine, glycine and glucose. Analysis of PLS-DA 1D loadings plot express high level of TAG in CC whereas higher levels of amino acids, beta-hydroxybutyrate, lactate, alanine, lysine, choline compounds, creatine, taurine, glucose, glycine etc. were detected in GBC as similarly observed in PCA model. For validation of the generated model, discriminating between benign (CC, XGC) and malignant (GBC) was done using the remaining 25 % of the sample (CC; n = 15, XGC; n = 6, GBC; n = 6), which were predicted using this PLS-DA model. Unsupervised prediction was performed and it demonstrated more than 95 % prediction were correct (Fig. 5b) when compared with histo-pathological ndings. 3.4 Semi-quantitative analysis

Uridine Uracil

Unknown

8.0

7.5
Taurine Choline compounds

7.0

6.5

6.0
TAG TAG

5.5 ppm

TAG

Myo-inositol Glycine

Myo-inositol

TAG

Glucose

Creatine

Lactate

Lysine

4.5

4.0

3.5
1

3.0

Aspartic Acid

2.5

2.0

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Alanine Lactate

1.0

Val, Leu, Ile

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TAG

TAG

TAG

ppm

Fig. 2 Stack plot of typical H HR-MAS NMR spectra of a, d GBC, b, e XGC, and c, f CC tissues showing difference in the metabolic prole

3.3 PLS-DA analysis of CC, XGC and GBC NMR spectra PLS-DA analysis was also performed for better characterization of the metabolites distinguishing CC, XGC and GBC groups from each other. PLS-DA was performed on the 1D CPMG HR-MAS spectra obtained from gall bladder tissues associated with CC, XGC and GBC. The full cross validated scores plot showed statistically signicant differences between CC and GBC while few of the XGC samples overlapped with GBC (Fig. 5a) as observed in PCA analysis. The PLS-DA model provided 100 % sensitivity and 94.12 % specicity. Discrimination between the benign and malignant tissues was due to TAG, BCA,

The above PCA/PLS-DA analysis performed using the integral area of a particular bin divided by the integral area of whole binned spectral region used for binning which demonstrate it as a relative method. Therefore it may or may not be able to reect absolute concentration of metabolites in each particular group i.e. CC, XGC and GBC. For example relative intensity of glucose in GBC samples was found higher as already represented in PCA and PLS-DA loading plots, whereas absolute intensity is lower as compared to CC and XGC (Fig. 6). For estimation of intensity of metabolites, QUANTAS signal with a xed intensity and line width at desired chemical shift was added in all the spectra individually (Bharti and Roy 2012). Mean integral area with standard error has been shown in Fig. 6 for lipids and small molecules. Statistical signicance for metabolites using their integral area was determined by Man-Whitney U test and detailed report has been provided in Supplementary Table S-2. The entire lipid components including TAG and cholesterol varied signicantly in decreasing order from CC to XGC and GBC. Small molecules such as taurine, glycine, glucose, choline containing compounds, creatine, uracil, tyrosine, amino acids etc. also vary from CC to XGC and GBC. Few resonances (at 4.60 (broad singlet), 5.60 (multiplet), 5.90 (triplet), 6.30 (triplet) ppm) which remained unassigned and their corresponding metabolites of these resonances could not be identied. However their absolute intensities in CC, XGC and GBC samples vary signicantly. Quantitative estimations of intensity of these resonances were also performed and their respective bar plots are also shown in Fig. 6.

4 Discussion This study demonstrates that CC samples were mostly dominated by TAG resonances whereas GBC samples were

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GBC

9.0

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Histopathological Examination

Fig. 3 Representative 800 MHz proton CPMG NMR spectra of CC, XGC and GBC tissues. a Routine histopathology photographs of the tissue specimens from same patients alongside of each NMR

spectrum. b The corresponding histopathology of the post HR-MAS NMR tissues are also shown which signies similar ndings when compared with a

dominated by small molecules like amino acids, choline, creatine, lactate, etc. The lipid prole of gall bladder tissues followed CC [ XGC [ GBC trends as observed by HR-MAS NMR which supports earlier study on lipids extract of gall bladder tissues (Jayalakshmi et al. 2011). Intensity of cholesterol C-18 CH3 signal was quantied with respect to QUANTAS signal which also represent same trends as TAG (Fig. 6). The detailed investigation of PCA and PLS-DA loadings/coefcients plots indicate high content of choline containing compounds (choline, choline, etc.), creatine, amino acids, lactate, glycine, taurine and b-hydroxybutyrate in GBC cases as compared to benign (CC and XGC) ones. Relative content of glucose with respect to total spectral area also varied signicantly among the groups. Lipids are the main source of fuel in mammalian cells for production of new cells. Carcinoma cells exhibit faster growth of cells where rate of energy expenditure is higher than its production results in utilization of stored lipids. Consequently lipid depletion occurs in cancer cells (McAndrew 1986). Hence lower content of lipid (TAG) was observed in GBC as compared to CC samples. However the similar results for lipid depletion in cancer cells of oral, breast, liver, etc. have been reported by HR-MAS NMR spectroscopy (Sitter et al. 2009; Srivastava et al. 2011). This study demonstrates signicant variation in TAG and cholesterol content among CC, XGC and GBC. Depletion in the cholesterol level may be attributed to the production of cholesterol ester in GBC. However, we were not able to authenticate the identication of the cholesterol ester signal at 4.60 ppm, but its presence as a broad

multiplet was observed in GBC spectra (Fig. 6) and showed a signicant increase in its level when compared with other groups. The absolute intensity of lactate could not be quantied due to overlap of TAG resonance at 1.33 ppm and TAGGlycerol at 4.13 ppm. However, the loading proles of PCA and PLS-DA models have been recently used to estimate the relative levels of lactate in malignant cells (Cao et al. 2012). Similarly, in our study, the PCA loading plot demonstrates increase in relative lactate concentration in GBC samples. It may be attributed to high glycolytic rate in malignant cells. Ischemia may develop as samples were outside for 510 min during surgical removal of the gallbladder. Glycolysis produces NADH which is oxidized by the mitochondria, but during ischemia or hypoxia conditions, this oxidative route becomes nonfunctional (Lane and Gardner 2005). Thus, in the ischemic tissue, conversion of pyruvate to lactate is the only way of oxidizing cytosolic NADH (Sitter et al. 2002). Therefore, during this period little contribution from ischemia and anaerobic glycolysis to lactate concentration may affect its actual tissues concentration (Tessem et al. 2008). Glucose to lactate conversion also protects cancer cells from oxidative stress (due to reactive oxygen) resulting in reduction of glucose level in cancer cells (McFate et al. 2008) as compared to benign and non-involved tissues (Gribbestad et al. 1994). The absolute intensity of glucose as shown in Fig. 6 showed minor decrease from CC to XGC to GBC, but no statistical signicance could be deciphered from analysis of the data. A proper explanation for this unusual observation could not be ascertained. However, strong

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0.06 0.04

PC-3 (4%)

0.02 0.00 -0.02 -0.04 -0.06 0.15 -0.05 0.00 0.10 0.05 0.05 0.00 -0.05 -0.10 -0.15 0.10 PC-1 (78% -0.20 -0.10

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-5

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Tyrosine

Glycine

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-10 9 8

-0.2 6 5 4 3 2 1

PC-3 (4%)

Fig. 4 A Three dimensional score plot derived using PC1m PC2 and PC3 from PCA analysis of 1H HR-MAS CPMG NMR spectra of CC, XGC and GBC tissues. B One dimensional principal component (a) PC1, (b) PC2, and (c) PC3 loadings plot derived from PCA analysis of CC, XGC and GBC tissues 1H HR-MAS CPMG NMR spectra. The water region from 4.7 to 5.1 ppm have been omitted in all the spectra during the binning procedure and hence shown as dotted straight line in the loading plots

signal of b-hydroxybutyrate in GBC samples was observed. Ketone bodies like b-hydroxybutyrate, acetone, etc. are produced in cancer cells under oxidative stress conditions (Pavlides et al. 2010). Presence of b-hydroxybutyrate may be an indication of oxidative stress in GBC tissues. Alanine, which is also an indication of hypoxic condition in cancer cells, was high in GBC tissues. Similarly, relative and absolute increased content of glycine was also observed in GBC samples. Increase in the lactate signal accompanied with glycine and alanine is probably due to increased rate of glycolysis in GBC tissues. Elevation in the levels of amino acids like valine, lysine, etc. were observed in GBC samples, again implying the involvement of glycolysis (Yang et al. 2007). One of the most robust indicator and widely used biomarker of malignant cells is signicant elevation in the choline containing compounds involved in membrane phospholipid metabolism, cell signaling, lipid transport etc. reported in several malignant tissues of different organs like brain, oral, breast, prostate etc. (Beloueche-Babari et al. 2009; Glunde et al. 2006; Srivastava et al. 2011). In sequence with previous published reports, choline containing compounds were also high in GBC samples as compared to CC and XGC. Both absolute and relative intensities were high in GBC representing active cell proliferation in cancer tissues. It is one of the biomarker which also used as in vivo MRS in clinical practices (Bolan et al. 2003). Taurine, important in osmoregulation and volume regulation process, also helps in protecting cells from swelling and free radical under hypoxic and oxidative stress conditions (Grifn and Shockcor 2004; Shen et al. 2001). Taurine reported as a potential diagnostic biomarker in differentiation of malignant from benign and control tissues and its level was found to be increased (Sitter et al. 2010; Wang et al. 2010; Srivastava et al. 2011). In GBC samples, relative as well as absolute intensities of taurine were signicantly increased. Three broad signals at 5.60, 5.90 and 6.30 ppm could not be assigned and were predominantly observed in CC and XGC samples, whereas it was not detected in any of the GBC tissue specimens (Fig. 6). The characteristization of these signals may provide biological correlation for discriminating malignancy. Whereas, uracil, an integral component of nucleotide/nucleoside was observed in XGC and GBC samples, it was not detected in CC samples. It is reported that nucleotide/nucleoside like ATP/ADP, UTP/UDP-hexoses, NAD, etc. involved in the energy metabolism, increases in malignant cells as compared to benign and control (Gribbestad et al. 1994). In line with this hypothesis, nucleotide/nucleoside signals resonating between 8.1 and 8.3 ppm were integrated with respect to QUANTAS signal. Elevation in their level was found to increase sequentially from CC \ XGC \ GBC samples (Fig. 6).

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Formate

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Glucose

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TAG TAG

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Alanine Lactate BHB BCA BC

Fumaric Acids

Formate Nucleotide

Glucose

PC -2

(1 1%

Metabolic prole of gall bladder tissues by HR-MAS NMR Fig. 5 a PLS-DA cross validated score plot derived using regression coefcient 1, 2 and 3 from PLS-DA analysis of CC, XGC and GBC 1H HRMAS CPMG NMR spectra. b Prediction of unknown gall bladder tissues using PLS-DA model which was prepared using training data set (CC; n = 51, XGC; n = 15 and GBC; n = 19) samples. This model was then used to predict the unknown (CC, XGC and GBC) samples. The predictions are made on the basis of a priori cut-off value of 0.5 and 1.5 for class membership, using y-predicted box-plot (class 0 for CC, 1 for XGC and class 2 for GBC). The predicted mean values along with standard deviation are depicted. All samples except one XGC sample denoted by * were correctly predicted by the training set model

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Factor-3 (3%, 19%)

10 5 0 -5 -10 -15 -40 10 -20 0 0 20

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Samples

CC and XGC are both inammatory conditions usually associated with gallstones but the factors that triggers CC in one patients and XGC in another is still not clear. It is also not known whether XGC represents an evolution of and later stage of CC. The major problem in diagnosis of XGC from GBC in clinical practice is mimicking GBC characteristics by XGC samples in physical appearance, wall thickening and similar image on diagnostic imaging techniques and may coexist with GBC (Makino et al. 2009; Ghosh et al. 2011; Karabulut et al. 2003). The individual analysis of NMR spectra showed that metabolic prole of some XGC samples mimics GBC metabolic prole. Other XGC samples had metabolic prole similar to CC samples that is higher TAG content as compared to GBC samples. It showed high content of TAG in XGC samples as compared to GBC. This may be attributed to accumulation of lipidladen macrophages in the area of destructive inammation which is a characteristic feature of XGC (Karabulut et al. 2003). In case of XGC, more than 70 % of the samples were predicted correctly by PLS-DA model and classied

as benign samples i.e., in CC groups The association of XGC with GBC is controversial but several articles have reported repetitively on the association of XGC with malignancy (Benbow 1989; Benbow and Taylor 1988; Ghosh et al. 2011; Goodman and Ishak 1981; Karabulut et al. 2003; Krishnani et al. 2000; Lopez et al. 1991; Ros and Goodman 1997) along with its mimicking and coexistence with GBC (Benbow 1990; Dixit et al. 1998; Houston et al. 1994; Kim et al. 1999; Krishnani et al. 2000; Kwon and Sakaida 2007; Parra et al. 2000; Ros and Goodman 1997). In our study, XGC samples overlapped with GBC in PCA/PLS-DA analysis and showed similar metabolic prole with GBC indicating similar metabolic disturbances. The premalignant potential of XGC therefore remains controversial. However, quantitative estimation of metabolites in XGC showed intermediate values between CC and GBC as shown in Fig. 6. All the above pattern recognition based PCA/PLS-DA analyses were performed using the relative integral area approach. It is a well established method, applied frequently

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# Taurine
Mean Area SD

# Glycine

# Creatine

#
CC XGC GBC CC XGC GBC CC XGC GBC

# / Choline Contn. Comps.


Mean Area SD

# / Uracil

# Tyrosine

CC

XGC

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# Amino Acids 3.78 ppm


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# / Adenine/Adenosine

Glucose

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CC

XGC

GBC

CC

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* / # Unknown 6.30 ppm


Mean Area SD

* / # Unknown 5.60 ppm

* / # / Unknown 5.90 ppm

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GBC

CC

XGC

GBC

CC

XGC

GBC

Unknown 4.60 ppm


Mean Area SD

* / # / Cholesterol

* / # TAG: 4.31 ppm

CC

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GBC

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* / # / TAG 0.90 ppm


Mean Area SD

* / # TAG 2.80 ppm

* / # / TAG 5.30 ppm CC XGC GBC

CC

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XGC

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Metabolic prole of gall bladder tissues by HR-MAS NMR


b Fig. 6 Quantitative variations of lipids and small molecules in CC,

115

XGC and GBC samples represented as bar plot of absolute integral area normalized with respect to QUANTAS signal. Mean area is represented as bar plot and standard deviation as error bars. Symbols *, # and / denote signicant changes among CC versus XGC, CC versus GBC and XGC versus GBC respectively. The detailed statistical analysis is shown in Supplementary Table S-2 using median values

generated from PCA/PLS-DA analysis of CC, XGC and GBC samples, but the absolute intensity of glucose in GBC samples was found to be lower when compared with CC and XGC tissue specimens (Fig. 7). Therefore, QUANTAS signal can be used for scaling in such pattern recognition statistical analysis for evaluating the quantitative (not relative) information of metabolites.

in many areas of research discipline to remove or minimize the effects of variable dilutions, weight variations etc., has its advantage over other methods in which integral area of a bin is divided by sum of integral area of all bins (Craig et al. 2006). Positive loadings of principal components (PCA) or regression coefcients (PLS-DA) for metabolites in one group indicate its higher content/concentration in all samples of that group. It does not mean that absolute concentration of those metabolites will be high in samples of respective group. Suppose in NMR spectra of group A, metabolite M has its absolute integral area X and total spectral area is 1000X, then relative integral area used in PLS-DA/PCA using such approach will be 0.00X. In second group B, suppose M has same absolute area X but total absolute area of spectrum is 10X, therefore its nal relative area used for analysis using similar approach will be 0.X. Hence PCA/PLS-DA analysis between A and B will show positive loading for metabolites M in group B but the absolute concentration of metabolites M is same in both the groups. This is one of the advantages of this approach that it separates group on the basis of relative integration with respect to the total spectral area but contrary does not reect the absolute concentration. For example, glucose showed positive loadings for GBC samples as observed in PCA and PLS-DA loadings plots

5 Conclusion The HR-MAS NMR spectroscopy is an adequate option for evaluating the metabolic prole of gall bladder tissues rather than extraction procedures which are time consuming, laborious and increase the risk of contamination. However, this is the rst study attempted by HR-MAS NMR based metabolic proling of small molecules as well as lipids and its implementation for differentiation of different pathology of gall bladder tissues i.e., CC, XGC and GBC. The CC samples could easily be distinguished from GBC samples using either small molecule metabolites or lipid resonances. Overlapping of 27 % of the XGC samples with GBC in the PLS-DA model projected it to be a more aggressive benign inammatory condition with rapid cell proliferation and this requires further investigation. XGC appears to be intermediate on a scale of metabolic changes from CC to GBC and the clustering of some of the XGC specimens with GBC may be a pointer to a later and perhaps premalignant stage of XGC. Though the number of patients in the present study is not too large and the results are preliminary. We believe that monitoring the metabolites observed in the present study could potentially be used in future as a diagnostic

A
0.0025 0.0020

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Fig. 7 a Mean value of relative intensity of glucose in CC, XGC and GBC has been shown. Relative intensity of glucose is dened as area of glucose signal is divided by area of the total spectrum (scaling used for PCA and PLS-DA analysis). b Mean value of absolute intensity of glucose has been calculated using QUANTAS and normalization was carried out with respect to area of QUANTAS signal. The bar diagram demonstrates that relative intensity of glucose has signicant

variation between CC and GBC a, therefore in PCA loading plot, glucose showed positive loading for GBC. However, absolute intensity of glucose as calculated by QUANTAS shows insignicant variations (Supplementary Table S-2). Therefore articial signal can be used for scaling the data and to estimate the quantitative information of metabolites

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S. K. Bharti et al. as, S. (2011). The Duportet, X., Aggio, R., Carneiro, S., & Villas-Bo biological interpretation of metabolomic data can be misled by the extraction method used. Metabolomics, 8(3), 410421. Elwood, D. R. (2008). Cholecystitis. Surgical Clinics of North America, 88(6), 12411252. Fisher, R. A., & Yates, F. (1957). Statistical tables for biological, agricultural, and medical research (5th ed.). Edinburgh: Oliver and Boyd. Ghosh, M., Sakhuja, P., & Agarwal, A. K. (2011). Xanthogranulomatous cholecystitis: A premalignant condition? Hepatobiliary & pancreatic diseases international, 10(2), 179184. Glunde, K., Jacobs, M. A., & Bhujwalla, Z. M. (2006). Choline metabolism in cancer: Implications for diagnosis and therapy. Expert Review of Molecular Diagnostics, 6(6), 821829. Goodman, Z. D., & Ishak, K. G. (1981). Xanthogranulomatous cholecystitis. American Journal of Surgical Pathology, 5(7), 653659. Gribbestad, I. S., Petersen, S. B., Fjosne, H. E., Kvinnsland, S., & Krane, J. (1994). 1H NMR spectroscopic characterization of perchloric acid extracts from breast carcinomas and noninvolved breast tissue. NMR in Biomedicine, 7(4), 181194. Grifn, J. L., & Shockcor, J. P. (2004). Metabolic proles of cancer cells. Nature Reviews Cancer, 4(7), 551561. Gupta, S. K., & Shukla, V. K. (2005). Gall Bladder cancer etiopathology and treatment. Health Administrator, 17(1), 134142. Hollywood, K., Brison, D. R., & Goodacre, R. (2006). Metabolomics: Current technologies and future trends. Proteomics, 6(17), 47164723. Houston, J. P., Collins, M. C., Cameron, I., Reed, M. W., Parsons, M. A., & Roberts, K. M. (1994). Xanthogranulomatous cholecystitis. British Journal of Surgery, 81(7), 10301032. ICMR. (1996). Annual report of population based cancer registries of the National Cancer Registry Programme (1993) (p. 18). New Delhi: ICMR Publication. Jayalakshmi, K., Sonkar, K., Behari, A., Kapoor, V. K., & Sinha, N. (2011). Lipid proling of cancerous and benign gallbladder tissues by 1H NMR spectroscopy. NMR in Biomedicine, 24(4), 335342. Jessurun, J., & Albores-Saavendra, J. (1996). Gallbladder and extrahepatic biliary ducts Vol. 2 andersons pathology. Saint Louis: CV Mosby. Kapoor, V. K. (2007). Advanced gallbladder cancer: Indian middle path. Journal of Hepato-Biliary-Pancreatic Surgery, 14(4), 366373. Karabulut, Z., Besim, H., Hamamci, O., Bostanoglu, S., & Korkmaz, A. (2003). Xanthogranulomatous cholecystitis. Retrospective analysis of 12 cases. Acta Chirurgica Belgica, 103(3), 297 299. Kim, P. N., Lee, S. H., Gong, G. Y., Kim, J. G., Ha, H. K., Lee, Y. J., et al. (1999). Xanthogranulomatous cholecystitis: Radiologic ndings with histologic correlation that focuses on intramural nodules. AJR. American Journal of Roentgenology, 172(4), 949953. Krishnani, N., Shukla, S., Jain, M., Pandey, R., & Gupta, R. K. (2000). Fine needle aspiration cytology in xanthogranulomatous cholecystitis, gallbladder adenocarcinoma and coexistent lesions. Acta Cytologica, 44(4), 508514. Kwon, A. H., & Sakaida, N. (2007). Simultaneous presence of xanthogranulomatous cholecystitis and gallbladder cancer. Journal of Gastroenterology, 42(8), 703704. Lane, M., & Gardner, D. K. (2005). Mitochondrial malate-aspartate shuttle regulates mouse embryo nutrient consumption. Journal of Biological Chemistry, 280(18), 1836118367. Lazcano-Ponce, E. C., Miquel, J. F., Munoz, N., Herrero, R., Ferrecio, C., Wistuba, I. I., et al. (2001). Epidemiology and molecular

bio-marker for gall bladder diseases using non-invasive volume localized in vivo MRS technique.
Acknowledgments Financial assistance from the Department of Science and Technology, Government of India is gratefully acknowledged. S. K. Bharti thanks Dr. K. Jayalakshmi Mulge and Ms Kanchan Sonkar for their help during the work.

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