Metabolomics (2012) 8:540555 DOI 10.

1007/s11306-011-0339-7

ORIGINAL ARTICLE

Metabolomic proling of amoebic and pyogenic liver abscesses: an in vitro NMR study
Santosh Kumar Bharti Virendra Jaiswal Ujjala Ghoshal Uday Chand Ghoshal Sanjay S. Baijal Raja Roy Chunni Lal Khetrapal

Received: 19 May 2011 / Accepted: 9 July 2011 / Published online: 16 July 2011 Springer Science+Business Media, LLC 2011

Abstract Pus samples obtained from 109 patients with liver abscess were examined by NMR spectroscopy. To our knowledge this is the rst report on metabolic proling of liver abscesses. Fifty metabolites were identied by combination of one (1D) and two-dimensional (2D) NMR spectra. Metabolic derangements were evaluated for differentiation between amoebic (ALA) and pyogenic liver abscess (PLA). The NMR results indicate that aspartate, asparagine and galactose, integral components of lipoproteophophoglycans (LPG) of the cell wall of Entamoeba histolytica are metabolic biomarkers of ALA. On the other hand, acetate, propionate, butyrate, succinate and formate, the fermentation products the facultative anaerobes are signicantly prevalent in PLA. The NMR based metabolic

prole of ALA and PLA are evaluated taking polymerase chain reaction (PCR) and bacterial culture as gold standard method. However, when NMR results were compared with culture and PCR methods, a correct diagnosis of 94.11% in ALA (n = 85) and 100% in PLA (n = 10) cases were observed. NMR spectroscopy in conjunction with PCR and culture can expedite in differentiating ALA from PLA. Keywords NMR spectroscopy Amoebic liver abscess Pyogenic liver abscess Metabolic proling PCR Culture Principal Component Analysis Abbrevations CPMG PCR TOCSY DQF-COSY HSQC

Santosh Kumar Bharti, Virendra Jaiswal are the authors have contributed equally.

Electronic supplementary material The online version of this article (doi:10.1007/s11306-011-0339-7) contains supplementary material, which is available to authorized users.
S. K. Bharti R. Roy C. L. Khetrapal (&) Centre of Biomedical Magnetic Resonance, Sanjay Gandhi Postgraduate Institute of Medical Sciences Campus, Raibarely Road, Lucknow, Uttar Pradesh 226014, India e-mail: clkhetrapal@hotmail.com V. Jaiswal U. Ghoshal Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India U. C. Ghoshal Department of Gastroenterology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India S. S. Baijal Department of Radiodiagnosis, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

CarrPurcellMeiboomGill Polymerase chain reaction Total correlation spectroscopy Double quantum ltered-correlation spectroscopy Heteronuclear single quantum coherence spectroscopy

1 Introduction Liver abscess is a collection of pus accumulated in a cavity formed by localised hepatic infection in the liver commonly caused by infection with protozoan parasite Entamoeba histolytica as amoebic liver abscess (ALA) or gut derived bacteria such as Escherichia coli, Klebsiella pneumoniae etc., as pyogenic liver abscess (PLA). Other types of liver abscesses like fungal, tubercular etc. are also found but rare or relatively less frequent (Huang et al. 1996; Rahmatulla et al. 2001). World Health Organization (WHO) estimates that amoebiasis is one of the three most common causes of death from parasitic diseases. Mortality

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mainly results from extra-intestinal infections, ALA being the commonest. Differentiation of ALA from PLA is important as management and outcome of the two conditions may differ (Lodhi et al. 2004). Clinical features and conventional laboratory parameters of the two conditions are similar and include fever, cough, right upper quadrant abdominal pain, leukocytosis, enlarged liver and raised right dome of the diaphragm on chest radiograph. There are some specic sonographic feature of ALA that differ from PLA but these differences are not sufcient for differentiation (Ralls et al. 1987). Demonstration of E. histolytica, the causative organism of ALA, on microscopy of pus sample is infrequent (Haque et al. 2000). Laboratory diagnosis of ALA is usually made by conventional serum antiamoebic antibody test. However, it cannot differentiate past infection from current infection and is also positive in a proportion of healthy population in endemic areas (Khairnar and Parija 2007). Detection of amoebic antigen has low sensitivity, particularly in patients partly treated with anti-amoebic drugs (Haque et al. 2000; Parija and Khairnar 2007; Zeehaida et al. 2008). Several polymerase chain reaction (PCR)-based methods have been developed to amplify DNA of E. histolytica in liver pus and stool specimens (Acuna-Soto et al. 1993; Khan et al. 2006; Tannich and Burchard 1991; WHO 1997). PCR is one of the methods having best sensitivity and specicity for detection of E. histolytica in liver pus samples as compared to other techniques (Fotedar et al. 2007a, b; Verweij et al. 2004) and also recommended by WHO (1997). The PCR is elegant technique and frequently adopted by biological scientists but routinely not applied in pathology laboratory because of its complexity and difcult procedures (Latchman 1995). PCR was chosen because of its high sensitivity for diagnosis of ALA and comparing the NMR metabolic prole. PLA is conventionally diagnosed by positive bacterial culture in liver pus in absence of anti-amoebic antibody in serum. However, sensitivity of this criterion is low if antibiotics have been used previously. The metabolomics approach to provide insight into their metabolic status and pathophysiology of pus formation has not been yet reported. On the other hand such an approach may provide desired information on ALA as well as PLA. Metabolomics allows the qualitative and quantitative measurement of all metabolites present in cell, biouids, pathological uids, tissue and tissue extract (Dunn and Ellis 2005; Lindon et al. 2000, 2003). The biochemical composition of ALAs and PLAs may differ. Among all the other analytical techniques used for metabolomics studies, high resolution NMR spectroscopy is widely used for investigating the composition of body uids, tissues extract, pathological uids, as a wide range of metabolites can be detected simultaneously without separation of individual components. Therefore, we hypothesize that metabolic prole of pus from liver

abscess would be different in patients with ALA and PLA. Accordingly we undertook this study with the following aims, (a) Metabolic proling of liver abscess, (b) To nd out the metabolic differences between ALA and PLA taking PCR and bacterial culture as gold standard methods.

2 Materials and methods 2.1 Subjects One hundred nine patients undergoing drainage of liver abscess in the Department of Radiodiagnosis in a tertiary care centre during a 3 year period (January 2006 to December 2009) were included in this study. Classication of liver abscesses was based on result of E. histolytica-PCR and bacterial culture which were previously described (Virendra Jaiswal et al. 2010). Patients diagnosed to have, mixed infection with E. histolytica and bacteria were excluded. Informed consent was obtained from all the patients and the protocol was approved by Institutions Ethics Committee (PGI/DIR/RC/957/2007). 2.2 Sample preparations for NMR Five ml of liver pus specimen was collected from each patient during drainage of abscess. An aliquot (approximately 2 ml) of liver pus was immediately stored in -80C for 1H NMR analysis and the remaining sample was processed microbiologically for another study, which has been reported previously (Virendra Jaiswal et al. 2010). Before performing the NMR analysis, samples were thawed at room temperature. The whole volume of sample was sonicated to homogenise and the centrifuge at 12,000 rpm for 10 min at 4C temperature to remove the suspended particulate matter. For 1H NMR experiments 250 ll of supernatant was pipetted out and makeup to 500 ll with deuterium oxide (D2O). For quantitative evaluation of various metabolites, sample was taken in 5 mm NMR tube with a Wilmad coaxial insert lled with known concentration of TSP (Sodium salt of 3-trimethylsilyl-(2,2,3,3-d4)propionic acid) in deuterium oxide. All the chemicals used for NMR analysis were purchased from Sigma Aldrich, USA. All the samples were coded and randomly provided for NMR analysis and PCR and Culture results were not disclosed until complete NMR analysis. 2.3 Experimental 1H NMR spectroscopy The NMR experiments were performed on a Bruker Avance 400 MHz spectrometer equipped with a 5 mm Broad Band Inverse probe shielded with z-gradients. During the analysis, sample temperature was 300 K. One dimensional (1D)

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single pulse experiments and CarrPurcellMeiboomGill (CPMG) sequence were used with presaturation pulse to suppress the water signal. The parameters used are: 8 kHz spectral width, 32K data points, 90 ip angle (10.6 ls), a total repetition time of 19.04 s and total echo time of 269 ms (for CPMG experiments), number of scan 128, dummy scan 8. The resultant spectra were processed using exponential line broadening of 0.3 Hz before Fourier transformation. Manual phase correction was performed followed by automatic base line correction. Unambiguous assignments of various metabolites were performed using 2D homonuclear and heteronuclear NMR spectroscopy. Some of the resonances were also conrmed by spiking experiments using standard compounds. The magnitude mode double quantum ltered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY) and 1 H13C heteronuclear single quantum coherence (HSQC) spectroscopy were performed for the assignments of resonances. For DQF-COSY 2K data points were collected in t2 domain over a spectral width of 8 kHz, water resonance was presaturated during relaxation delay of 2.0 s, 512 t1 increments with 64 transient scans and 16 dummy scans. For TOCSY, all basic parameters were same as in DQF-COSY. Spin lock time used was 70 ms. 512 increments were collected in t1 dimension using 88 transient scan. The resulting 2D data were Fourier transformed after zero lling in the t1 dimension to 1,024 points and multiplying in both dimension by sine bell window function. All data acquisition and processing were performed using XWINNMR 3.5 version. The parameters used for 1H13C HSQC were: 2K data points in t2 dimension over spectral width of 5,952 Hz, 500 t1 increments with 80 transients, relaxation delay of 2.0 s, acquisition time of 170 ms and 90 pulse length. The spectral width in t1 dimension was 20,124 Hz. The phase sensitive data were obtained by the antiecho-time proportional phase increments (Antiecho-TPPI) method. The resulting data were zero-lled to 512 data points and were weighted with 90 squared sine window functions in both the dimensions prior to Fourier transformation. 2.4 PCR and bacterial culture of pus Five ml of liver pus obtained during drainage of abscess was examined for bacteria by Grams staining and bacterial culture as per standard methods (Collee et al. 1996). PCR analysis was performed on the sample for the detection of E. histolytica in pus sample using Zaman et al. method (2000). 2.5 Statistical analysis Principal Component Analysis (PCA) on NMR Spectra: The CPMG spectra obtained from ALA (n = 85) and PLA

(n = 10) were subjected for the multivariate PCA. The spectra were reduced to 395 (between 0.5 and 9.0 ppm) discrete chemical shift regions by digitization to produce a series of sequentially integrated regions of 0.02 ppm width, using Bruker AMIX software (Version 3.8.7, Bruker GmbH, Germany). The region of 4.55.1 ppm was excluded from the analysis to remove the residual signal of HOD, water and distorted region due to water suppression. The data obtained was mean centered, scaled to Pareto Scaling and then normalized by dividing each integral 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 matrix was further subjected for the PCA. Univariate analysis of quantitative and categorical data: MannWhitney U test and Fisher Exact test (SPSS 11.5) was applied on the quantitative data and categorical data respectively.

3 Results Fourteen patients were excluded from the nal analysis as a denite diagnosis could not be made due to inadequate work-up (n = 3), tubercular liver abscess (n = 1), fungal liver abscess (n = 1), mixed infection (n = 9). Eighty-ve patients with ALA (85/104, 81.7%) and ten with PLA (10/104, 9.6%) were nally included in this study. Therefore the total sample size was from 95 patients. Male were more affected by ALA (88%) as compared to PLA (55%). Mean age of the ALA (41.1 15.6 years) and PLA (42.2 23.1 years) patients were almost same and no signicant differences were found in our study. The mean age of male and female in ALA group was 42.0 16.4 and 40.5 19.7 years respectively. 3.1 Identication of metabolites by NMR spectroscopy The NMR spectra of liver abscesses were analyzed by the combined use of 1D and 2D NMR spectroscopy. A typical 1D 1H NMR spectrum of ALA is shown in Fig. 1. The typical 2D COSY, 1H13C HSQC and TOCSY spectra used for assignments of the compounds are shown in Figs. 2, 3 and Supplementary Fig. 1 respectively with resonance assignments. The assignments were also carried out on the basis of coupling pattern, coupling constant and chemical shift reported in the literature (Bollard et al. 2009; Denkert et al. 2008; Foxall et al. 1993; Gao et al. 2009; Govindaraju et al. 2000; Grand et al. 1999; Gupta et al. 2001; Lai et al. 2005; Lindon et al. 1999; Nicholson et al. 1995; Nicholson and Wilson 1989; Pinero-Sagredo et al. 2010; Rocha et al. 2010; Silwood et al. 2002; Sweatman et al. 1993), comparison with reference compounds present in Biological

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Phenylalanine Tryptophan

543

Uracil / Tryptophan

Adenine

Tyrosine

Formate

Tyrosine

Uracil

UDP / Uridine

Tryptophan

Fumaric Acid

UDP / Uridine

6X

Galactose

Glucose

(B)
8.5 8.0 7.5 7.0 6.5 6.0 5.5 V aline/Leucine/Isoleucine Ethanol ppm

GABA / Lysine

Succinate

Glucose

Lactate

GABA

Galactose

Acetoacetate

GABA

Acetate

Taurine Choline

V aline

Taurine

Aspartic Acid Methionine

Asparagine

Threonine

4.6

Glutamine

Lysine

Lactate / Threonine

Glycine

Alanine

(A)
4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

Fig. 1 Typical 1D 1H NMR spectrum of ALA showing metabolite assignments. Expansion of the spectrum from a 0.54.8 ppm and b 5.09.0 ppm

Magnetic Resonance Data Bank and Human Metabolome Data Base (Markley et al. 2007; Wishart et al. 2009). The results of some of the spiking experiments (Supplementary Fig. 2) and details procedure for comparing standard 1D

(Supplementary Fig. 3), COSY (Supplementary Fig. 4), TOCSY (Supplementary Fig. 5), HSQC (Supplementary Fig. 6) spectra with liver abscess spectra are presented in supplementary materials. The 1H NMR spectra of liver pus

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Fig. 2 2D 1H1H COSY spectrum of ALA along with the assignment of the metabolites. (Ileu isoleucine, Leu leucine, Val valine, Lys lysine, Glu glutamate, Gln glutamine, Meth methionine, Arg arginine,

Eth ethanol, Pro proline, Ala alanine, Lac lactate, Thre threonine, Cit citrate, Asp aspartic acid, Asn asparagine, Tyr tyrosine, His histidine, Phe phenylalanine, Tau taurine, Try tryptophan, Gluc glucose)

samples were highly complex and it did not allow all the assignment on the basis of chemical shift reported in literatures. 2D homonuclear NMR experiments were performed to resolve the spectral complexity. Even resonance overlapping was also observed in COSY and TOCSY spectra in the chemical shift region of 3.04.25 ppm. Therefore 1H13C HSQC was recorded and it provided better resolution for assignments of metabolites due to more dispersion of chemical shift in 13C dimension.

Aspartic acid, asparagine and galactose were observed in low concentrations and therefore assigned by spiking experiments (Supplementary Fig. 2). Whereas, acetic acid, formic acid, succinate and acetoacetate were assigned by the 13C chemical shifts obtained from the HSQC spectra as well as by spiking with known standard compounds in the 1 H NMR spectra. Different classes of metabolites such as carbohydrates, organic acids, aliphatic and aromatic amino acids (AAA), bacterial fermentation products and lipids

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ppm
Ornithine Alanine Methionine Threonine Isoleucine Isoleucine V aline Proline Ornithine Glutamic Acid Proline Tryptophan Methionine

15 20

V aline Leucine Leucine Lactate Lysine Isoleucine Leucine Citrulline

25 30 35 40 45 50 55 60 65 70 75

Histidine V aline Asparagine Tyrosine Lysine Methionine Isoleucine Phenylalanine Tryptophan Histidine Asparagine Citrate Aspartic Acid Alanine Leucine Methionine Lysine Betaine Choline Proline Citrulline Aspartic Acid Lysine Ornithine Leucine Glutamic Acid Glycine

Ornithine Serine Tyrosine Phenylalanine Isoleucine V aline Glucose Glycerol Gluconic Acid Serine Choline Glycerol Glucose

Proline

Threonine Lactate Gluconic Acid Myo-Inositol Glucose Gluconic Acid Myo-Inositol

Glucose

Glucose Myo-Inositol Glucose

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

ppm

Fig. 3 2D 1H13C HSQC spectrum of ALA. The assignments of the metabolites are labelled

were detected in the 1H NMR spectra liver pus. The detailed 1H NMR chemical shift assignment of the metabolites observed in the liver pus samples are presented in Table 1. GABA was observed in very high concentration in some of the samples and it was assigned by TOCSY as shown in (Supplementary Fig. 1).

3.2 PCA of NMR spectra Multivariate analysis (PCA) was performed on the CPMG 1 H NMR spectra of ALA and PLA as it provides better baseline. A seven-component model explained 98.0% of the variance, with the rst two components explaining 93.5.0%

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546 Table 1 1H chemical shift assignments of the metabolites observed in the NMR spectra of ALAs and PLAs S. no. 1 2 3 4 5 Name of metabolites Acetic acidb Acetoacetate Adenine Alanine Arginine Chem. shift 1.92 2.23 (s) 8.19 (s) 8.21 (s) 1.48 (d) 3.78 (q) 1.71 (m) 1.91 (m) 3.25 (d) 3.77 (t) 6 Asparaginea 2.87 (dd) 2.95 (dd) 4.01 (dd) 7 Aspartic acida 2.69 (dd) 2.82 (dd) 3.90 (dd) 8 9 Betaine Beta-hydroxybutyrate 3.26 3.90 1.20 (d) 2.31 (dd) 2.43 (dd) 4.13 10 n-Butyric acidb 0.90 (t) 1.57 (m) 2.18 (t) 11 Choline 3.21 (s) 3.53 4.07 12 13 Citric acid Citrulline 2.53 (d) 2.67 (d) 3.75 3.15 (t) 1.88 (m) 1.57 (m) 14 15 16 17 18 19 20 21 Creatine Cysteine Dimethyl amine Ethanol Formic acidb Fumaric acid Galactosea GABA 3.03 (s) 3.93 3.06 3.94 2.75 (s) 1.18 (t) 3.62 8.46 (s) 6.51 (s) 4.60 (d) 1.90 (m) 2.30 (t) 3.02 (t) Resonances CH3 CH3 3CH/8CH 3CH/8CH b-CH3 a-CH c-CH2 b-CH2 d-CH2 a-CH b-CH b0 -CH a-CH b-CH b -CH a-CH NCH3 CH2 CH3 b-CH b0 -CH a-CH CH3 3CH2 2CH2 N(CH3)3 NCH2 OCH2 CH2 CH2 a-CH d-CH2 c-CH2 b-CH2 CH3 CH2 b-CH2 a-CH CH3 CH3 CH2 CH CH 1CH 3CH2 2CH2 4CH2
0

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Assignment methods 1D/HSQC/spiking 1D/spiking 1D 1D/COSY/HSQC 1D/COSY/HSQC

1D/COSY/HSQC/spiking

1D/COSY/HSQC/spiking

1D/HSQC 1D/COSY/HSQC

1D/TOCSY/HSQC

1D/HSQC

1D/COSY/HSQC 1D/TOCSY/HSQC

1D/HSQC 1D/HSQC 1D/HSQC/spiking 1D/COSY/HSQC 1D/HSQC/spiking 1D 1D/spiking 1D/COSY/HSQC

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Metabolic prole of pus from liver abscesses Table 1 continued S. no. 22 Name of metabolites a-Glucose Chem. shift 3.41 3.54 3.71 3.83 3.85 5.24 (d) 23 b-Glucose 3.24 3.41 3.47 3.49 3.74 3.91 24 Gluconic acid 4.65 (d) 4.11 4.02 3.81 3.65 3.75 3.75 25 Glutamate 2.09 2.35 (m) 3.77 26 Glutamine 2.13 (m) 2.45 (m) 3.77 27 28 Glycine Glycerol 3.56 3.57 3.66 3.79 29 Histidine 3.18 3.25 3.99 7.13 (s) 7.98 (s) 30 Isoleucine 0.94 (t) 1.01 (d) 1.26 (m) 1.47 (m) 1.98 (m) 3.68 (d) 31 32 Lactate Leucine 1.33 (d) 4.12 (q) 0.96 (d) 0.97 (d) 1.71 (m) 3.75 33 Lipids 0.90/0.96 1.29 Resonances C4H C2H C3H C6H C5H C1H C2H C4H C5H C3H C6H C60 H C1H 2CH 3CH 6CH2 6CH2 4CH 5CH b-CH2 c-CH2 a-CH b-CH2 c-CH2 a-CH CH2 1,3CH2 1,3CH2 2CH b-CH b0 -CH a-CH C4H-ring C2H-ring d-CH3 c-CH3 c-CH c0 -CH b-CH a-CH b-CH3 a-CH d-CH3 d0 -CH3 c-CH/b-CH2 a-CH CH3 (CH2)n 1D/COSY/TOCSY 1D/COSY/HSQC 1D/HSQC 1D/HSQC 1D/COSY/HSQC 1D/COSY/HSQC 1D/HSQC Assignment methods

547

1D/COSY/TOSCY/HSQC

1D/COSY/TOSCY/HSQC

1D/COSY/TOSCY/HSQC

1D/COSY/TOSCY/HSQC

1D/COSY/TOSCY/HSQC

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548 Table 1 continued S. no. Name of metabolites Chem. shift 1.58 2.04/2.07 2.25 2.81 5.32 34 Lysine 1.47 (m) 1.72 (m) 1.9 (m) 3.02 3.74 35 Methionine 2.13 (s) 2.16 (m) 2.64 (t) 3.85 36 Myo-inositol 3.28 (t) 3.54 (d) 3.62 (t) 4.06 (t) 37 Ornithine 1.81 (m) 1.92 (m) 3.05 (t) 3.78 38 Phenylalanine 3.12 3.28 3.98 7.32 (d) 7.37 (m) 39 40 Propionic acidb Proline 7.41 (m) 1.05 (t) 2.18 (q) 2.01 (m) 2.08 (m) 2.35 (m) 3.35 3.42 4.13 41 42 43 44 45 Serine Succinateb Scyllo-inositol Taurine Threonine 3.85 (m) 3.97 (m) 2.46 (s) 3.35 (s) 3.25 (t) 3.41 (t) 1.34 (d) 3.6 (d) 4.25 (m) 46 Tryptophan 3.29 (dd) 3.47 (dd) Resonances CH2CH2CO CH=CHCH2 CH2CO CH=CHCH2CH=CH CH=CH c-CH2 b-CH2 d-CH2 NCH2 a-CH SCH3 b-CH2 c-CH2 a-CH C2H-ring C1,3H-ring C5H-ring C4,6H-ring c-CH2 b-CH2 d-CH2 a-CH b-CH b0 -CH a-CH C2H, C6H-ring C4H-ring C3H, C5H-ring CH3 CH2 c-CH2 b-CH b0 -CH d-CH d0 -CH a-CH b-CH2 a-CH CH2 CH SCH2 NCH2 c-CH3 a-CH b-CH b-CH b0 -CH

S. K. Bharti et al.

Assignment methods

1D/COSY/TOSCY/HSQC

1D/COSY/HSQC

1D/COSY/HSQC

1D/COSY/HSQC

1D/COSY/HSQC

1D/COSY/HSQC 1D/COSY/HSQC

1D/COSY/HSQC 1D/HSQC/spiking 1D/HSQC 1D/COSY/HSQC 1D/COSY/HSQC

1D/COSY/HSQC

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Metabolic prole of pus from liver abscesses Table 1 continued S. no. Name of metabolites Chem. shift 4.04 (dd) 7.19 (t) 7.26 (t) 7.30 (s) 7.53 (d) 7.72 (d) 47 Tyramine 2.92 (t) 3.23 6.88 (d) 7.21 (d) 48 Tyrosine 3.06 (dd) 3.19 (dd) 3.95 (dd) 6.89 (d) 7.18 (d) 49 50 Uracil Valine 5.8 (s) 7.54 (d) 0.99 (d) 1.04 (d) 3.62 (d) 2.28
a b

549

Resonances a-CH C5H-ring C6H-ring C2H-ring C4H-ring C7H-ring CH2 NCH2 3,5CH 2,5CH b-CH b0 -CH a-CH C3H, C5H-ring C2H, C6H-ring C5H-ring C6H-ring c-CH3 c0 -CH3 b-CH a-CH

Assignment methods

1D/COSY/HSQC

1D/COSY/HSQC

1D/COSY/HSQC 1D/COSY/TOCSY/HSQC

Metabolites specic to ALA and metabolites specic to PLA. All other metabolites were common in both ALA and PLA samples

ALA (n=85) PLA (n=10)

1.5 1.0

PC-3

0.5 0.0 -0.5 -1.0 1.0 0.5 0.0 -0.5 -0.5 0.0 0.5 1.5

1.0

PC-2

-1.0 -1.5 -2.0 -2.5 -2.0

-1.5

-1.0

PC

-1

Fig. 4 PCA of CPMG 1H NMR spectra of ALA and PLA. Scores plot PCA discriminating ALA from PLA based on selected metabolites. ALA cases mark by circle, have acetate, succinate and formate in the NMR spectra

asparagine, aspartic acid, glucose, branch chain amino acids (BCA), AAA, lactate, alanine etc. (Fig. 5). The analysis of 2D loading plot showed that PLA samples were separated from ALA mainly due to acetate, succinate and formate (Supplementary Fig. 7). Dispersion in the ALA group was observed due variations in BCA, AAA, lactate, aspartic acid, asparagine, alanine etc. One of the ALA sample was overlapped with PLA group (Fig. 4) and detail spectral analysis of this sample showed high content of acetate, succinate and formate which are key metabolites in PLA. Aspartic acid and asparagine were also present in this sample but their intensities were relatively low when compared with acetate, succinate and formate, proving their major role in separating PLA from ALA. Acetate, succinate and formate were also observed in two other ALA samples but in less concentration and therefore classied in the cluster of ALA group (Fig. 5). 3.3 NMR spectral assignments, quantitation and statistical analysis The stack plots of the two representative PLA and ALA pus samples 1H NMR spectra are shown in Fig. 6 so as to highlight the major differences in the metabolites. The absence of asparagine, aspartic acid and galactose resonances was very straightforward in the PLA spectrum.

of the total variance. The clear clustering between ALA and PLA in the PCA of spectra demonstrated signicant metabolic variations in ALA and PLA groups (Fig. 4). Examination of PC1, PC2 and PC3 loadings showed that the cluster separation arising mainly due to acetate, succinate, formate,

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Water Region

Alanine

0.2

0.0

-0.2
Lactate Lactate

-0.4 9.0 8.0 7.0 6.0 5.0

Formate

AAA

4.0

3.0
Asparagine Aspartic Acid

2.0

Acetate

1.0

Alanine

0.4

PC-2 Loading
Glucose AAA

0.2

0.0
Formate

-0.4 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0

-0.4

PC-3 Loading
AAA Lactate Lactate BCA

-0.2

0.0
Glucose

-0.2

-0.4 9.0 8.0 7.0 6.0 5.0 4.0

Glycine

3.0

2.0

Acetate

-0.2

Succinate

1.0

Fig. 5 PCA of CPMG 1H NMR spectra of ALA and PLA. 1D loading plot of a PC 1, b PC 2 and c PC 3 generated from PCA shown in Fig. 4. PC 1 and PC 2 are the major component for group separation

While in ALA, absence of acetate, propionate, butyrate, succinate and formate signals were observed. A doublet at 4.60 ppm arising due to anomeric proton signal of galactose was observed only in *40% ALA pus samples. A known concentration of TSP was used in coaxial insert to avoid the quantitative error arising due to interaction of TSP with proteins present in the sample. A relaxation time of 19.04 s was given to ensure the full relaxation of all resonances. Relative integral of metabolites resonances were normalise to TSP signal. Concentration of the TSP present in coaxial insert was validated by known concentration of glycine before quantication of the metabolites (Larive et al. 1997). Quantitation of only 17 metabolites was performed from patients with ALA and PLA. However, quantitation of propionate and butyrate could not be performed due to

BCA

BCA

0.4

Succinate

PC-1 Loading

overlapping of the resonances with other signals. Quantitative variability of the common metabolites was found to be statistically insignicant between ALA and PLA (Table 2). The presence of aspartic acid, asparagine and/or galactose was found to be ngerprint metabolites in ALA cases, while acetate, succinate, propionate, butyrate and formate were only observed in PLA cases. Sample size in PLA group was quite low as compared to ALA. Therefore, in order to verify the bacterial fermentation products specic to PLA, an in vitro study has been performed by incubating bacterial clinical isolates (K. pneumoniae) in bacteria free liver abscess (ALA) having no previous fermentation product as observed by NMR spectroscopy. K. pneumoniae was chosen because it the most common isolates of PLA (Chang et al. 2000; Cheng et al. 1989; Lee et al. 2001; Rahimian et al. 2004). The ALA sample mixed with K. pneumoniae suspension in phosphate buffered saline and incubated for 18 h at 37C. Detail procedure and results are reported in supplementary material. After 18 h incubation liver abscess sample showed bacterial fermentation products acetate, formate, succinate, propionate etc. in very high concentration (Supplementary Fig. 8) as already reported in all the 10 PLA samples. Butyrate and propionate which are specically found in PLA did not demonstrated signicant role in PCA clustering of spectra. This may due to their low concentration and overlapping with other signals. Similarly, a doublet signal at 4.60 ppm for galactose was found to be insignicant in the PCA loading plots when tried with including galactose region. This may due to its very low concentration and was observed in only *40% of the samples from ALA. Therefore, it was thought worthwhile that detailed analysis of individual spectra followed by univariate analysis may help for better discrimination. On the basis of these distinct resonances, ALA and PLA were classied and compared with PCR and culture results and the details are presented in (Table 3). The data presented is quite intriguing, the ALA samples can be 94.11% correctly classied by NMR. Three patients had bacterial fermentation products in their pus samples along with asparagine and aspartic acids detected by NMR spectroscopy. All the ten PLA samples were correctly identied by NMR spectroscopy. Two ALA patients could not be classied by NMR spectroscopy as it neither showed bacterial fermentation products nor aspartic acid and asparagine in their pus samples.

4 Discussion The results of 1H NMR spectroscopy of liver pus specimens provide evidence that the metabolic prole of ALA is different from that of PLA. Unsupervised multivariate PCA also clearly demonstrated the differential metabolic prole

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Aspartic Acid Aspartic Acid Methionine

551

Asparagine

Asparagine

(C)

Glucose

Glucose

Asp / Asn

3.0

2.9

2.8

2.7

2.6

(B)
Succinate Acetate Ethanol

Formate

(A)
8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 ppm

Fig. 6 A stack plot of typical 1H NMR spectra of a PLAs and b ALAs showing difference in the metabolic prole. c Expansion of the spectra between 2.6 to 3.0 ppm

of ALA and PLA. Even the NMR based metabolic prole of ALA was found to be different when compared with other pyogenic abscesses like brain abscesses and abdominal abscesses (Grand et al. 1999; Lai et al. 2002, 2005). This indicates that metabolism of E. histolytica is entirely different from bacterial metabolism. One of the characteristic features of E. histolytica is the presence of cell wall rich surface antigens, lipoproteophophoglycans (LPG), rich in aspartic acid, serine, glutamic acid, glucose and galactose (Sue Moody-Haupt 2000). The presence of aspartic acid and galactose in ALA may therefore be attributed to the constituent of the cell wall of the E. histolytica in the pus samples. Another plausible source of aspartic acid, asparagine and galactose may be related to its specic metabolism or due to different mode of pathogenesis of E. histolytica (http://www.genome.jp/kegg-bin/show_pathway? ehi00052) which is entirely different from the bacterial metabolism (Anderson and Loftus 2005). In the absence of glucose E. histolytica utilizes several amino acids such as asparagine, aspartate, threonine, tryptophan, methionine, homocysteine etc. for generation of ATP (Anderson and

Loftus 2005). Proton NMR spectra of ALA suggests that glucose was present in a considerable amount and thus amino acids were not utilized by E. histolytica. However, glucose utilization by E. histolytica is lower as compared to bacteria as conrmed by quantitative analysis (Table 2). The metabolism predicted on the basis of genome sequence of E. histolytica also suggests the production of ethanol and propionate (Loftus et al. 2005). Ethanol was observed in amoebic liver pus but not specic to ALA samples only. Whereas, propionate was observed along with acetate and succinate in PLA samples indicating end products of the bacterial fermentation. Metabolic prole of PLA showed additional resonances such as acetate, succinate, propionate, formate and butyrate and absence of aspartate, asparagine and galactose. These metabolites present in the PLA are well known fermentation product of bacterial metabolism (Clark 1989; Murarka et al. 2008; Wang et al. 2010). Our results of in vitro fermentation liver abscess with K. pneumoniae also substantiate that succinate, acetate, formate, propionate etc. are the end product of bacterial fermentations. The presence of

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S. K. Bharti et al.

Table 2 Evaluation of statistical signicance of metabolites quantied and specic metabolites as observed in the 1H NMR spectra of ALA and PLA Metabolites Tryptophan Phenylalanine Histidine Tyrosine Uracil Glucose Threonine Valine Glycine Lysine Alanine Lactate Acetate Succinate Formate Aspartic Acid Asparagine Galactose Propionate Butyrate ALA (mg/dl) (n = 85) Median (range) 1.88 (0.0029.62) (70%)a 16.32 (0.00145.40) (98.82%) 10.18 (0.0077.96) (95.3%) 12.73 (0.00101.92) (98.82%) 0.67 (0.008.74) (62.35%) 30.90 (0.00442.06) (97.64%) 23.34 (150.10) (97.64%) 22.52 (0.00141.62) (98.82%) 12.35 (0.1978.88) (100%) 81.69 (1.28628.88) (100%) 26.59 (0.43157.42) (100%) 82.82 (7.65319.90) (100%) 0.00 (2.1032.08) (3.52%) Not detected (3.52%) (1.9528.0) Not detected (3.52%) (1.1012.34) 10.24 (0.4064.44) (100%) 3.74 (0.0026.23) (98.82%) Not quantied (38.50%) Not detected (0.00%) Not detected (0.00%) NS* not signicant i.e. P values [0.05
a

PLA (mg/dl) (n = 10) Median (range) 1.94 (0.0017.92) (80%) 9.39 (1.10114.66) (100%) 4.66 (0.0024.94) (90%) 6.68 (0.7663.60) (100%) 0.48 (0.0015.04) (90%) 10.63 (0.0077.56) (80%) 10.38 (0.66103.18) (100%) 8.83 (1.40156.58) (100%) 5.23 (1.0095.14) (100%) 52.23 (6.00747.00) (100%) 11.92 (1.66198.42) (100%) 60.54 (9.30293.16) (100%) 32.23 (4.34130.72) (100%) 5.10 (0.95155.13) (100%) 6.83 (1.5317.77) (100%) Not detected (0.00%) Not detected (0.00%) Not detected (0.00%) Not quantiedb (100%) Not quantied (100%)

P values NS* NS NS NS NS NS NS NS NS NS NS NS 0.001 0.001 0.001 0.001 0.001 0.014 0.001 0.001

Percentage denes the presence of metabolites in respective group. Example: Tryptophan was present in the 70% of the ALA i.e. in 30% ALA sample it was absent. bNot quantied means metabolites detected in respective group but we are unable to quantify because of signal overlap

their strong signal in liver abscess spectra clearly indicates the abscess with pyogenic origin. Earlier, in vivo and in vitro 1H NMR investigations had revealed the presence of

acetate and succinate as bacterial fermentation product in the spectra of pyogenic brain abscesses. These signals had been used as a diagnostic biomarker for the differentiation

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553

Table 3 Classication of NMR results on the basis of distinct metabolic prole taking PCR and bacterial culture methods as gold standard method Gold standard method (N = 95) NMR method ALA PCR ?ve, B.Cult. -ve PCR -ve, B.Cult. ?ve ALA (n = 85) PLA (n = 10) 80 0 PLA 0 10 Mixeda 3 0 Sterileb 2 0

NMR results were classied on the basis of aspartic acid, asparagine/galactose in ALA and bacterial fermentation product such as acetate, succinate, formate, butyrate and propionate in PLA. Gold Standard Criteria: ALA; PCR positive for E. histolytica and culture negative for bacteria and PLA; PCR negative for E. histolytica and culture positive for bacteria. Other cases were excluded as discussed in Sect. 2
a b

Mixed (both ALA and PLA) was classied on the basis of NMR metabolic prole Neither ALA nor PLA was classied on the basis of NMR metabolic prole

of pyogenic abscesses from tumors and tuberculous brain abscesses (Garg et al. 2004; Grand et al. 1999; Gupta et al. 2001; Kim et al. 1997; Lai et al. 2002, 2005). These fermentation products had also been utilized for the monitoring of treatment. (Burtscher and Holtas 1999). Therefore, the presence of strong signals of acetate, succinate, formate, and propionate in liver pus samples clearly demonstrate the presence of pyogenic infection. Three ALA cases were classied as mixed infection by NMR, the plausible reason may be the patients had received antibiotic treatment prior to drainage of liver abscess. Hence, their culture results were sterile but their fermentation metabolites persisted in pus samples. NMR spectroscopy was unable to classify two ALA cases and this is may be because of low concentration of asparagine and aspartate which was beyond the NMR detection limit. Moreover, bacterial fermentation products were also not observed in these cases, indicating absence of bacterial infection. Therefore, large sample size is required to ensure the role of these metabolites for differentiation in liver abscess especially in mixed infection.

fungal, tubercular etc., verication of metabolic prole of such abscesses is required using NMR spectroscopy.
Acknowledgment Financial assistance from the Department of Science and Technology, Govt. of India and Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India are gratefully acknowledged.

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