Combined NMR and GCâMS Analyses Revealed Dynamic Metabolic

Article pubs.acs.org/jpr

Combined NMR and GC−MS Analyses Revealed Dynamic Metabolic Changes Associated with the Carrageenan-Induced Rat Pleurisy Huihui Li,† Yanpeng An,†,‡ Lulu Zhang,† Hehua Lei,† Limin Zhang,† Yulan Wang,†,§ and Huiru Tang*,†,‡ †

Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Centre for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, University of Chinese Academy of Sciences, Wuhan 430071, P. R. China ‡ State Key Laboratory of Genetic Engineering, Biospectroscopy and Metabolomics, School of Life Sciences, Fudan University, Shanghai 200433, P. R. China § Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, P. R. China S Supporting Information *

ABSTRACT: Inflammation is closely associated with pathogenesis of various metabolic disorders, cardiovascular diseases, and cancers. To understand the systems responses to localized inflammation, we analyzed the dynamic metabolic changes in rat plasma and urine associated with the carrageenan-induced self-limiting pleurisy using NMR spectroscopy in conjunction with multivariate data analysis. Fatty acids in plasma were also analyzed using GC−FID/MS with the data from clinical chemistry and histopathology as complementary information. We found that in the acute phase of inflammation rats with pleurisy had significantly lower levels in serum albumin, fatty acids, and lipoproteins but higher globulin level and larger quantity of pleural exudate than controls. The carrageenaninduced inflammation was accompanied by significant metabolic alterations involving TCA cycle, glycolysis, biosyntheses of acute phase proteins, and metabolisms of amino acids, fatty acids, ketone bodies, and choline in acute phase. The resolution process of pleurisy was heterogeneous, and two subgroups were observed for the inflammatory rats at day-6 post treatment with different metabolic features together with the quantity of pleural exudate and weights of thymus and spleen. The metabolic differences between these subgroups were reflected in the levels of albumin and acute-phase proteins, the degree of returning to normality for multiple metabolic pathways including glycolysis, TCA cycle, gut microbiota functions, and metabolisms of lipids, choline and vitamin B3. These findings provided some essential details for the dynamic metabolic changes associated with the carrageenaninduced self-limiting inflammation and demonstrated the combined NMR and GC−FID/MS analysis as a powerful approach for understanding biochemical aspects of inflammation. KEYWORDS: carrageenan-injection, pleurisy, inflammation, metabonomics, fatty acids

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INTRODUCTION

Acute inflammation normally resolves although the mechanisms have remained somewhat elusive so far. It is now believed that an actively coordinated program of resolution generally initiates soon after inflammatory responses with biosynthesis of lipoxins promoted from the arachidonate-derived prostaglandins and leukotrienes.1 This coincides with the biosynthesis of resolvins and protectins from n-3 polyunsaturated fatty acids to initiate the termination sequence by shortening the neutrophil infiltration period and apoptosis.1 Subsequently, phagocytosis of apoptotic neutrophils (by macrophages) results in neutrophil clearance accompanied by release of anti-inflammatory and reparative cytokines.1 However, prolonged inflammation (also

Inflammation is a part of the complex biological responses of an organism’s immune system and vascular tissues to harmful stimuli such as pathogens and injurious physical insults.1 Inflammation can be normally classified into the acute and chronic phases.1 The former is the initial response of the body to these stimuli with the increased movement of plasma and leukocytes (especially granulocytes) to the injured tissues. Under such circumstances, a cascade of biochemical events will propagate involving the immune and local vascular systems together with a variety of cells in the injured tissue.1 Although the typical signs for acute inflammation include swelling, heat, redness, and pain, inflammation is actually a protective attempt to fight against these stimuli and usually to initiate a healing process. © 2013 American Chemical Society

Received: May 8, 2013 Published: October 17, 2013 5520

dx.doi.org/10.1021/pr400440d | J. Proteome Res. 2013, 12, 5520−5534

Journal of Proteome Research

Article

clearly illustrated some detailed associations of inflammatory processes with arachidonate metabolism. The responses of glycolysis and Krebs cycle to the DSS-induced ulcerative colitis were observed,39 and the depletion of short-chain fatty acids in fecal extracts was also reported for patients with inflammatory bowel diseases.29 This suggests that more metabolic pathways in systems level are probably responsive to inflammation development and progression because arachidonic acid metabolism is not isolated from other fatty acids and the whole metabolic network. However, so far, only limited studies have been reported for studying the inflammatory effects on the whole metabolic network using the metabonomic analysis given the importance of inflammation. No report has been published so far on the metabolic phenotypes of mammals with the carrageenaninduced pleurisy to the best of our knowledge. We analyzed the dynamic metabolic changes in rat blood plasma and urine samples resulting from the carrageenaninduced pleurisy using NMR. Fatty acid composition in blood plasma was also analyzed with GC-FID/MS techniques as a function of the inflammation development. The objectives of this work are to understand the rat metabonomic responses to the development of carrageenan-induced pleurisy and reversibility of such responses during the self-limiting inflammatory process.

known as chronic inflammation) causes simultaneous injuries of tissues due to prolonged destruction of cell integrity leading to many diseases, such as metabolic disorders,2 cardiovascular disease,3,4 or even cancer.5,6 Therefore, inflammation is normally closely regulated by the body, and the mechanistic aspects of inflammation with animal models are important research topics. Carrageenan-induced pleurisy is one of the most widely used acute inflammatory models used in understanding the inflammation and in development of anti-inflammatory drugs.7 Carrageenan is a family of linear sulfated polysaccharides from marine algae,8 and its injection to rats causes pleurisy, which is characterized with the increased pleural exudates and alterations to biosynthesis of prostaglandins.9 Under such conditions, “acute phase” responses for inflammation occur with some dramatic concentration changes for various acute phase proteins10 in blood plasma so as to prevent tissue damages and to activate the repairing processes.11 This acute phase reaction is initiated and mediated by a number of cytokines, such as IL-1β, IL-6, and NFκB, with inflammatory activities from a variety of cell types including leucocytes, lymphocytes, and monocytes.12,13 These inflammatory cytokines in different tissues are involved in amplification and regulatory pathway controlling the development of acute phase response in vivo.12,13 With the progression of inflammatory processes, inflammation either becomes selflimited and finally resolved or proceeds to a low-grade chronic state. It is now well known that among the inflammatory mediators, nitric oxide (NO) and prostaglandins play important roles in the development of inflammatory diseases.14 Pro-inflammatory prostaglandins such as PGE2 are generated from arachidonic acid by constitutive and inducible cyclooxygenases15 in the early acute phase, whereas cyclopentenone prostaglandins are mainly produced16 in the resolution stage. In contrast, NO is produced from arginine by endothelial and neural constitutive and inducible NO synthases, suggesting important roles of oxidative stress in pathogenesis.17 This indicates that metabolisms of some fatty acids and amino acids are closely involved in inflammation development and progression at least at the inflammatory sites. However, it remains to be fully understood how the metabolic processes other than arachidonate and arginine are involved in inflammatory processes. Metabonomic analysis ought to be useful to investigate such biochemistry aspects of inflammation because metabonomics can holistically detect the metabolite composition of a biological system and its dynamic responses to both endogenous and exogenous stimuli.18,19 As a powerful approach, the NMR-based metabonomics has been successfully employed in characterizing the metabolic phenotypes of mammalian gastrointestinal tracts20−23 and understanding the molecular aspects of metabolic diseases.24−28 The metabonomic analysis has also been applied in investigating pathogenesis and progression of the inflammatory bowel diseases29,30 and parasitic diseases31−33 together with some lung diseases.34,35 More recently, targeted metabolomic analysis showed that LPS-induced systems inflammation was accompanied by clear changes of arachidonic acid metabolism involving COX, LOX, and P450 pathways.36 It has also been found that rofecoxib exposure caused drastic level increases of 20-HETE, which might be related to the rofecoxib-caused adverse cardiovascular events.37 Arachidonic acid stimulation caused obvious changes in biosynthesis of dozens of bioactive lipids such as prostaglandins and leukotrienes within the signaling cascade in chronic lymphocytic leukemia (CLL) cells.38 These studies

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MATERIALS AND METHODS

Chemicals

NaCl, K2CO3, K2HPO4·3H2O, NaH2PO4 ·2H2O, methanol, and hexane (all in analytical grade) were purchased from Sinopharm Chemical Reagent (Shanghai, China). Acetyl chloride, methyl heptadecanoate, and methyl tricosanate (99.0%) were obtained from Sigma-Aldrich (St. Louis, MO). D2O (99.9% D) and sodium 3-trimethylsilyl [2,2,3,3-2H4] propionate (TSP) were obtained from Cambridge Isotope Laboratories. 3,5-Di-tertbutyl-4-hydroxytoluene (BHT) and a standard for mixed methyl esters of 37 fatty acids was bought from Supelco (Bellefonte, PA). λ-Carrageenan was obtained from LTK Laboratories (St. Paul, MN). Phosphate buffer (PB) containing 0.01% NaN3 (w/ v) prepared from K2HPO4 and NaH2PO4 was used as a solvent for NMR analysis of urine and blood plasma samples due to its good stability at low temperature.40 Animal Experiments and Sample Collection

Animal experimental procedures were performed according to the National Guidelines for Experimental Animal Welfare (MOST of P. R. China, 2006). Forty-four male Sprague− Dawley rats (202.45 ± 9.44 g, 6 weeks old) were purchased from Vital River Laboratories (Beijing, China) and housed in an SPF animal laboratory with a 12 h light/dark cycle at a constant temperature of 20−25 °C and relative humidity of 40−60%. All animals had free access to water and normal food. After 2 weeks of acclimatization, the animals were randomly divided into two groups, namely, control and treated groups. Following light anaesthetization with isoflurane, the animals in the treated group were injected with 0.3 mL of λ-carrageenan suspension in sterile saline solution (1% wt/wt) in the right pleural cavity,7 whereas control rats received only saline injection. Under such conditions, the treated rats will develop an acute pleurisy, which is self-limiting and will finally resolve. Two days after injection, 11 rats in each group were sacrificed by neck dislocation under isoflurance anesthesia, and the remaining rats were sacrificed 6 days after carrageenantreatment. Pleural exudate from each animal was harvested by washing the pleural cavity with 1 mL of saline. The exudate 5521



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volumes were measured and expressed by subtracting the saline volumes. Blood samples were collected from retro-orbital vein plexus. Serum samples were prepared in a standard manner and used for clinical chemistry and GC−FID/MS analyses; plasma samples for NMR spectroscopic analysis were also prepared in a standard fashion with sodium heparin as anticoagulant. Other samples were also collected including urine (at day 0, 2, 4, and 6 post-treatment) and thymus and lung tissues (at day 2 and 6 post-treatment). All samples were immediately snap-frozen in liquid nitrogen after collection and stored at −80 °C until further analyses. Immediately after collection, lobes of lung and thymus were also, respectively, fixed in 10% formalin solution for histopathological assessments. Rat body weights were measured three times, namely, at the time point of pretreatment, day 2, and day 6 post-treatment.

For resonance assignment purposes, a series of 2D NMR spectra were acquired and processed as previously reported41−43 for selected samples. These included 1H−1H correlation spectroscopy (COSY), 1H−1H total correlation spectroscopy (TOCSY), 1H J-resolved spectroscopy (JRES), 1H−13C heteronuclear single quantum correlation spectroscopy (HSQC), and 1H−13C heteronuclear multiple bond correlation spectroscopy (HMBC). The levels for polyunsaturated fatty acids (PUFAs), unsaturated fatty acids (UFAs), and total fatty acids (ToFAs) were calculated from the diffusion-edited spectra as previously reported25 by using the signal integrals at δ 5.247−5.361 (for UFAs), δ 2.711−2.847 (for PUFAs), and δ 0.803−0.907 (for ToFAs, CH3 groups).

Clinical Chemistry and Histopathological Assessments

All of these 1-D 1H NMR spectra were corrected manually for possible phase and baseline distortions. Chemical shifts were referenced to the anomeric proton of α-glucose (δ 5.23) for plasma, whereas they were referenced to TSP (δ 0.00) for urine spectra. The regions of δ 0.5−8.5 for plasma and δ 0.5−9.0 for urine spectra were uniformly integrated into bins with the width of 0.004 ppm (i.e., 2.4 Hz) using AMIX package (v3.8, Bruker Biospin). The regions at δ 4.50−5.20 and δ 5.40−6.00 were discarded to eliminate the effects of imperfect water suppression and to remove urea signal. Data were then normalized to the sample volumes for plasma, whereas they were normalized to the sum of spectral integrals for urine to compensate the intersample concentration differences. Multivariate data analysis was carried out with software SIMCA-P+ (v.12.0, Umetrics, Sweden). Principle component analysis (PCA) was performed on mean-centered data to generate an overview of sample distribution and to observe possible outliners. The orthogonal projection to latent structure discriminant analysis (OPLS-DA) was performed to obtain the metabolites having significant contributions to intergroup differentiation with the Pareto-scaled NMR data as X-matrix and class information as Y-matrix. All OPLS-DA models were calculated with seven-fold cross-validation and further validated with CV-ANOVA at the level of p < 0.05. After back-transformation,44 loadings plots were generated for OPLS-DA models using an in-house developed MATLAB (V7.1, Mathworks, USA) script with each variable (or metabolite signal) color-coded by the correlation coefficients. In these loadings plots, the warm-colored (e.g., red) variables (or metabolites) had more significant contributions to intergroup differentiation than cold-colored (e.g., blue) ones. Cut-off values of correlation coefficients were determined depending on sample numbers based on the discrimination significance of the Pearson’s product-moment correlation coefficients at the level of p < 0.05.44

Data Processing and Multivariate Data Analysis

Clinical chemistry analyses were performed with a HITEC Integra 7080 automatic analyzer (Hitec, Hitachi, Japan) including total protein (TP), albumin (ALB), globulin (GLO), total cholesterol (tChol), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), glucose (Glc), and uric acid (UA). These data were expressed in the form of “mean ± standard deviation” and analyzed with the Student’s t test. Histopathological assessments were performed with the standard procedures. In brief, the formalin-fixed lung and thymus tissues were embedded in paraffin wax, sectioned (3 to 4 μm), and then stained with the hematoxylin and eosin (H&E) method. This and microscopic assessments were all conducted as a paid service by a local qualified pathologist. Sample Preparation for NMR Spectroscopy

Plasma samples were prepared by mixing 200 μL of plasma with 400 μL of phosphate buffer (45 mM, pH 7.4) containing 10% D2O. After vortexing and 10 min of centrifugation (14489 g, 4 °C), 550 μL of supernatant was transferred to a 5 mm NMR tube for each sample, followed by NMR analysis. Urine samples were prepared by mixing 500 μL of urine with 50 μL of phosphate buffer (1.5 M in D2O, pD 7.4) containing 0.05% TSP.40 Following 10 min of centrifugation (14 489 g, 4 °C), 500 μL of supernatant was transferred to a 5 mm NMR tube for each sample, followed by NMR analysis. NMR Measurements

All NMR spectra were acquired at 298 K on a Bruker Avance III 600 MHz NMR spectrometer equipped with an inverse cryogenic probe (Bruker Biospin, Germany). For plasma, three 1D spectra were acquired with NOESYPR1D, Carr−Purcell− Meiboom−Gill (CPMG), and diffusion-edited pulse sequences, respectively, as previously described.21−23 Water signal was saturated by employing a weak continuous wave irradiation during recycle delay (2 s) and mixing time (90 ms) in the case of NOESYPR1D. For urine samples, 1-D 1H NMR spectra were acquired using the first increment of the gradient-selected NOESY pulse sequence (NOESYGPPR1D) with water signal suppressed during both the recycle delay (2 s) and mixing time (80 ms). 90° pulse length was adjusted to ∼10 μs for each sample. Sixty-four transients were collected into 32 k data points over a spectral width of 20 ppm (i.e., 12 kHz). All free induction decays from the above experiments were multiplied by an exponential function with a line-broadening factor of 1 Hz and zero-filled to 128 k data points prior to Fourier transformation.

GC−FID/MS Analysis of Fatty Acids in Blood Plasma

Plasma fatty acids were methylated using a previously reported method45 with some modifications. In brief, 20 μL internal standards (1 mg/mL methyl heptadecanoate and 0.5 mg/mL methyl tricosanate) in hexane containing 3,5-di-tert-butyl-4hydroxytoluene (BHT) were added to a Pyrex tube, followed by the addition of 50 μL of rat plasma and 2 mL of methanol-hexane mixture (4:l v/v). After the sample tubes were cooled over a self-made liquid nitrogen bath for 15 min, 200 μL of precooled acetyl chloride was added to the above mixture. Following a brief nitrogen gas flush, tubes were then screw-capped and kept at room temperature in 5522



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Figure 1. Histopathological assessments of thymus and lung (stained with HE, Mag ×200) from control and carrageenan-injected rats: (A) thymus from control; thymus from carrageenan-treated rats (B) at day 2 and (C) at day 6 post-treatment; (D) lung from control; and lung from carrageenan-treated rats (E) at day 2 and (F) at day 6 post-treatment.

Table 1. Data for Serum Clinical Chemistry and Phenotypes from the Carrageenan-Induced Pleurisy Rats and Controlsa TP (g/L) ALB (g/L) GLO (g/L) A/G HDL-C (mmol/L) LDL-C (mmol/L) TG (mmol/L) tChol (mmol/L) Glc (mmol/L) UA (mmol/L) thymus weight (g) spleen weight (g) pleural exudate (mL)

control (d2)

pleurisy group (d2)

control (d6)

P6R

P6L

59.69 ± 2.30 32.77 ± 0.81 26.92 ± 1.74 1.20 ± 0.04 1.00 ± 0.08 0.25 ± 0.05 1.02 ± 0.44 2.29 ± 0.27 10.38 ± 0.70 49.03 ± 20.07 0.54 ± 0.12 0.68 ± 0.11 0.0 ± 0.0

60.97 ± 2.32 31.46 ± 0.79c 29.51 ± 1.66c 1.07 ± 0.04d 0.87 ± 0.06d 0.36 ± 0.12e 0.71 ± 0.21 2.16 ± 0.35 10.03 ± 1.10 55.15 ± 19.82 0.61 ± 0.19 0.69 ± 0.08 4.6 ± 2.0d

63.02 ± 2.88 35.23 ± 0.79 27.79 ± 2.15 1.27 ± 0.08 0.94 ± 0.08 0.21 ± 0.05 1.11 ± 0.32 1.90 ± 0.24 10.47 ± 1.92 70.93 ± 23.23 0.47 ± 0.11 0.65 ± 0.03 0.0 ± 0.0

61.11 ± 2.60 33.95 ± 0.81d 27.16 ± 1.96 1.25 ± 0.07 0.93 ± 0.07 0.22 ± 0.07 1.82 ± 0.21d 1.94 ± 0.25 10.93 ± 1.37 48.76 ± 30.95 0.41 ± 0.07 0.60 ± 0.13 0.0 ± 0.0

61.58 ± 2.56 34.20 ± 1.21e 27.38 ± 1.49 1.25 ± 0.04 0.97 ± 0.05 0.24 ± 0.05 0.91 ± 0.17 1.99 ± 0.16 10.78 ± 1.10 71.00 ± 44.32 1.12 ± 0.57e 0.57 ± 0.06e 0.8 ± 1.5

b

a

d2: day 2 post-treatment; d6: day 6 post-treatment; P6R and P6L: better and less resolved subgroups, respectively, at day 6 post-treatment. TP: total proteins; ALB: albumin; GLO: globulins; A/G: albumin-to-globulin ratio; HDL: high-density lipoproteins; LDL: low-density lipoproteins; TG: triglycerides; tChol: total cholesterol; Glc: glucose; UA: uric acid. bMean ± standard deviation. cp < 0.01. dp < 0.001. ep < 0.05.

25 °C/min. The temperature was kept at 205 °C for 3 min then increased to 225 °C (10 °C/min) and kept at 225 °C for 3 min. The MS spectra were acquired with the EI (70 eV) source and the m/z range of 45−450. Fatty acids were identified by comparing with a chromatogram from a mixture of 37 standards and further confirmed with their mass spectrometry data from standard libraries. Each fatty acid was quantified with the FID data by computing the signal integrals of each analyte and internal standards. The results were expressed as micromole fatty acids per liter of plasma in the form of “mean ± standard deviation”. From the above results, the molar percentages were calculated for saturated fatty acids (SFAs), UFAs, monounsaturated fatty acids (MUFAs), and PUFAs, respectively. The amounts of n3 and n6 types of PUFAs were also calculated. The inflammation-caused changes for metabolites were calculated against their levels in controls as, (Ct − Cc)/Cc, where Ct and Cc were the mean concentrations in the treated and control groups, respectively. Dynamic changes of metabolites were also investigated as a function of durations following carrageenan injection.

the dark for 24 h. The resultant mixture was cooled in an ice-bath for 10 min and then neutralized with dropwise addition of 5 mL of 6% K2CO3 solution (with gentle shaking). The mixture was then rested in the ice bath for 30 min, followed by the addition of 200 μL of hexane (to extract lipids). Following another 10 min of resting, the top layer was collected and transferred to a sample vial. The previous extraction process was further repeated twice, and the resultant three supernatants from each sample were combined and evaporated to dryness. So-obtained extracts were then carefully redissolved in 50 μL of hexane for GC−FID/MS analysis. Methylated fatty acids were measured on a Shimadzu GC2010Plus GC-MS spectrometer (Shimadzu Scientific Instruments, USA) equipped with a flame ionization detector (FID) and a mass spectrometer having an electron impact (EI) ion source. A DB-225 capillary GC column (10 m, 0.1 mm ID, 0.1 μm film thickness) from Agilent Technologies was employed with helium as carrier and makeup gas. Sample injection volume was 1 μL with a splitter (1:60). The injection port and detector temperatures were 230 °C. The column temperature was started from 155 °C for 1 min and then increased to 205 °C with a rate of 5523



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Figure 2. 1H NMR spectra for the plasma (A) and urine samples (B) from control and carrageenan-treated rats. The dotted regions were vertically expanded for clarity.

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RESULTS

indicating the heterogeneous resolution within the inflammation group. This heterogeneity was also confirmed from hierarchical cluster analysis of our NMR data (Figure S2 in the Supporting Information). However, no significant differences were observed for animal body weights between two groups throughout the period. Clinical chemistry data showed that in the acute phase of pleurisy, rats with inflammation had significantly lower levels of serum HDL, albumin, and albumin-to-globulin ratio but higher levels in globulin and LDL than controls (Table 1). In contrast, 6 days after treatment (i.e., in the resolution phase), both P6R and P6L subgroups still had lower serum albumin level than controls though HDL, and LDL and globulin levels returned to the normal levels for controls. Furthermore, the P6R subgroup showed higher triglyceride level than controls. In contrast, thymus weights and pleural exudate volumes were higher but spleen weights were lower in the P6L subgroup than in controls.

Phenotypic and Clinical Chemistry Assessments of Animal Models

Histopathological examinations showed that both interstitial hemorrhage and polymorphonuclear neutrophil accumulation were clearly observable in the lung of carrageenan treated rats (Figure 1). For thymus, the treated rats also had polymorphonuclear neutrophil accumulation in the connective tissue capsule and septa, whereas no significant changes were observable in the cortex and medulla (Figure 1). Six days after treatment, only a few polymorphonuclear neutrophil remained observable in both lung and thymus tissues (Figure 1). Results for pleural exudate volumes (Table 1) showed that our carrageenan injection successfully induced pleurisy with an acute phase and resolution phase.7 In the acute phase of inflammation (2 days after treatments), the carrageenan-injected rats had about 2−8 mL pleural exudate per rat, whereas little such fluid was obtained from the control rats (Figure S1 in the Supporting Information). Six days after treatment, most rats in the treated group showed no pleural exudate except for two animals (Figure S1 in the Supporting Information), which had about 2 to 3 mL of such liquid. This together with the thymus and spleen weights (Figure S1B in the Supporting Information) showed the presence of two subgroups (n = 6 each) in pleurisy group, namely, better resolved (P6R) and less resolved (P6L),

1

H NMR Spectra of Rat Plasma and Urine Samples

Typical 1H NMR spectra for rat plasma and urine samples (Figure 2) from both control and pleurisy groups showed rich information for endogenous metabolites. The resonances of these metabolites were comprehensively assigned (Table 2) according to literature data,46−48 publically accessible and inhouse databases, which were further confirmed individually with a series of 2D NMR spectral data. It is apparent that the abundant 5524



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Table 2. 1H and 13C NMR Data for Plasma and Urinary Metabolites from the Rat Pleurisy Model keys

metabolites

1 2 3

formate cholesterol lipid

4

valine

5

leucine

6

isoleucine

7

3-hydroxybutyrate

8

lactate

9

threonine

10

alanine

11

lysine

12 13 14 15

acetate N-acetyl-glycoproteins O-acetyl-glycoproteins arginine

16 17

acetoacetate glutamate

18

glutamine

19

4-PYc

moieties

δ 1H (multiplicitya)

δ 13C

CH CH3 CH3 CH3 (n3 fatty acid) CH2 CH2CH2CO CH2CC CH2CO CCCH2CC CHCH γCH3 γ′CH3 βCH αCH δCH3 δ′CH3 βCH2 γCH αCH δCH3 β′CH3 γCH2 γCH2′ βCH αCH γCH3 αCH2 αCH2′ βCH βCH3 αCH γCH3 αCH βCH βCH3 αCH γCH2 δCH2 εCH2 αCH CH3 NCH3 OCH3 γCH2 βCH2 δCH2 αCH CH3 βCH2 γCH2 αCH βCH2 γCH2 αCH 3-CH 2-CH 6-CH CO CO NCH3 3-CH

8.46(s) 0.80(s) 0.80(s) 0.86(t) 1.27(m) 1.57(m) 2.01(m) 2.23(m) 2.76(m) 5.30(m) 1.00(d) 1.05(d) 2.28(m) 3.62(d) 0.96(d) 0.97(d) 1.70(m) 1.72(m) 3.73(t) 0.94(t) 1.02(d) 1.26(m) 1.47(m) 1.99(m) 3.68(d) 1.20(d) 2.32(dd) 2.42(dd) 4.15(m) 1.33(d) 4.11(q) 1.32(d) 3.58(d) 4.24(m) 1.49(d) 3.77(q) 1.49(m) 1.74(m) 3.03(t) 3.76(t) 1.93(s) 2.07(s) 2.13(s) 1.70(m) 1.93(m) 3.25(t) 3.77(t) 2.28(s) 2.07(m) 2.35(m) 3.77(m) 2.17(m) 2.46(m) 3.75(m) 6.70(d) 7.83(dd) 8.56(d)

172.0 # 16.7 16.7 32.4 27.6 29.5 35.9 28.3 131.0 19.6 20.9 31.9 63.2 24.2 24.7 26.9 42.7 56.8 14.2 17.8 27.6 27.6 39.1 62.4 24.4 49.6 49.6 68.6 23.0 71.3 22.3 63.1 68.2 19.2 53.3 24.5 29.5 42.6 55.4 26.1 24.9 23.2 26.5 30.5 43.3 57.3 32.3 29.9 35.9 59.3 29.8 33.9 57.2 123.4 146.8 149.2 180.9 170.9 47.6 55.7

5525

3.90(s) 4.74(t)

samplesb U P P

P

P

P

P

P P

P P

P, U P P P

P P

P

U



Article

Table 2. continued keys

metabolites

20

proline

21

α-ketoglutarate

22 23 24

pyruvate succinate trigonelline

25

aspartate

26

methionine

27

citrate

28

dimethylglycine

29 30

dimethylamine guanine

31

creatinine

32

asparagine

33

serine

34

choline

35

phosphorylcholine

36

glycerophosphocholine

37

betaine

38

taurine

39 40

glycine glycerol

41 42

TMAO β-glucose

moieties γCH2 βCH2 βCH2′ δCH2 δCH2′ αCH αCH2 βCH2 COOH CO CH3 CH2 1-CH 2 or 4-CH 3-CH βCH2 βCH2′ αCH S-CH3 βCH2 γCH2 αCH CH2 CH2′ NCH3 CH2 CH3 CH CNH2 CC CC CH3 CH2 CH2 CH2′ αCH αCH βCH2 βCH2′ NCH3 OCH2 NCH2 NCH3 NCH2 OCH2 NCH3 NCH2 OCH2 CH3 CH2 SCH2 NCH2 CH2 CH2 CH2′ CH N−CH3 2-CH 4-CH 5-CH 3-CH


δ 13C

2.01(m) 2.07(m) 2.36(m) 3.34(m) 3.45(m) 4.14(m) 2.44(t) 3.01(t)

26.5 31.8 31.8 49.0 49.0 64.0 32.5 40.4 184.5 208.5 29.2 37.3 148.0 148.0 130.7 39.3 39.3 54.5 16.6 32.7 31.6 56.8 46.0 46.0 46.6 62.4 37.4 145.5 81.8 156.4 168.5 39.2 56.2 37.4 37.4 54.1 59.5 62.7 62.7 56.9 70.3 58.5 56.9 69.5 61.1 56.9 68.9 62.5 56.4 69.2 50.3 38.5 44.5 # # # 62.2 77.2 72.6 78.9 78.8

2.37(s) 2.41(s) 9.13(s) 8.84(dd) 8.08(d) 2.68(dd) 2.82(dd) 3.91(m) 2.14(s) 2.16(m) 2.65(t) 3.87(t) 2.69(d) 2.81(d) 2.92(s) 3.71(s) 2.72(s) 7.69(s)

3.04(s) 4.06(s) 2.84(dd) 2.94(dd) 3.98(m) 3.84(dd) 3.95(dd) 3.98(dd) 3.21(s) 3.53(t) 4.07(t) 3.23(s) 3.60(t) 4.17(t) 3.23(s) 3.69(t) 4.33(t) 3.27(s) 3.91(s) 3.27(t) 3.42(t) 3.57(s) 3.56(dd) 3.65(dd) 3.77(m) 3.27(s) 3.26(dd) 3.41(dd) 3.47(m) 3.50(t) 5526

samplesb P

U

P, U P, U U

P

P

P, U P, U U U

U P

P

P, U

P, U

P

P U P P

U P



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Table 2. continued keys

metabolites

43

α-glucose

44

triglycerides

45

creatine

46 47 48 49

methylamine allantoin urea NMNA

50

uridine

51

3-indoxyl sulfate

52

phenylacetylglycine

53 54

fumarate tyrosine

55

histidine

56

1-methylhistidine

57

phenylalanine

58

hippurate

59

unknown 1

moieties 6-CH 6-CH′ 1-CH 4-CH 2-CH 3-CH 6-CH 5-CH 1-CH OCH2 OCH2′ OCH CH2 CH3 CH3 CH NH2 CH3 5-CH 6-CH 4-CH 2-CH CHCH CHCH 4-CH 2-CH 5-C 5-CH 6-CH 2-CH 7-CH 4-CH CH2 2-CH 4,6-CH 3,5-CH C CO CH 3,5-CH 2,6-CH 2-CH 4-CH 2-CH 4-CH 2,6-CH 4-CH 3,5-CH α-CH2 3,5-CH 4-CH 2,6-CH CO COOH CH3 CH2


δ 13C

3.73(dd) 3.90(dd) 4.66(d) 3.42(dd) 3.54(dd) 3.71(dd) 3.78(m) 3.84(m) 5.24(d) 4.06(m) 4.26(m) 5.20(m) 3.03(s) 3.93(s) 2.61(s) 5.40(s) 5.79 4.48(s) 8.19(dd) 8.91(dt) 8.97(d) 9.29(s) 6.39(d) 7.31(d) 7.36(s) 7.86(s)

63.7 63.7 98.9 72.6 74.5 75.7 75.0 74.6 95.1 64.0 68.3 72.2 58.9 # 25.7 66.2 106.8 52.2 131.3 147.5 150.6 148.4 133.4 124.6 132.5 136.9 123.3 122.1 # # # 119.6 45.5 133.0 119.3 133.0 145.5 167.8 138.0 118.9 133.7 120.2 144.6 118.5 138.8 132.3 130.3 131.9 47.2 132.5 135.5 130.0 173.3 180.0 19.7 29.7

7.21(dd) 7.28(dd) 7.36(s) 7.51(d) 7.71(d) 3.65(s) 7.36(m) 7.36(m) 7.42(m)

6.52(s) 6.91(d) 7.20(d) 7.01(s) 7.70(s) 7.06(s) 7.81(s) 7.33(m) 7.38(m) 7.43(m) 3.97(s) 7.56(dd) 7.64(t) 7.83(dd)

1.21(t) 2.65(m)

samplesb

P, U

P

p U P, U U U

U

U

U

U P P P P

U

U

a s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets. bP, plasma; U, urine; # not determined. cTMAO: trimetlylamine oxide; 4-PY: N1-methyl-4-pyridone-3-carboxamide; NMNA: N-methylnicotinamide.

plasma metabolites include “acute phase” proteins including Nacetyl-glycoproteins (NAG) and O-acetyl-glycoproteins (OAG),

amino acids (e.g., Leu, Ile, Val, Ala, Lys, Phe, and Tyr), TCA cycle intermediates (citrate and succinate), ketone bodies 5527



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Figure 3. OPLS-DA scores plots (left) and loadings plots (right) derived from plasma data for control and carrageenan-treated rats. (A) Day 2 posttreatment and (B) day 6 post-treatment (subgroup P6R). Metabolite keys are given in Table 2

in the P6L than in P6R groups (Figure S3 in the Supporting Information). Loadings plots revealed that rats with pleurisy had significant differences in some plasma metabolites in the acute phase (Figure 3A). This is highlighted with higher levels for lysine, histidine, phenylalanine, NAG, OAG, lactate, and succinate together with lower levels for acetoacetate, cholesterol, and UFAs in the carrageenan-treated group (Figure 3A). In the resolution phase of inflammation, rats in the P6R subgroup showed significant higher levels for lipids (including some n3 type fatty acids), NAG, and OAG than controls (Figure 3B). No significant differences were observable for the plasma levels of small molecules including amino acids, TCA cycle intermediates, and lactate between this subgroup and controls (Figure 3B). In contrast, the OPLS-DA model for P6L and controls did not pass the rigorous test from CV-ANOVA, although the Q2 was greater than 0.4. Nevertheless, the Student’s t test results indicated that rats in the P6L subgroup had significantly higher plasma levels for NAG and tyrosine but lower levels in lactate, citrate, and glutamine than these in the control group (Figure S4 in the Supporting Information). Data from the other two types of NMR spectra (NOESYPR1D and diffusion-edited spectra) showed similar results to the above findings in terms of the carrageenan-induced changes in OAG and NAG. The results calculated from the diffusion-edited spectra25 (Figure S5 in the Supporting Information) indicated that carrageenan-induced significant level declines for PUFAs (δ 2.711−2.847), UFAs (δ 5.247−5.361), and ToFAs (at δ 0.803− 0.907), although the details for the fatty acid composition were not readily available from these spectra.

(acetone, 3-hydroxybutyrate, and acetoacetate), glycolysis products (pyruvate and lactate), choline metabolites (choline, phosphocholine, and glycerophosphocholine), and lipids (Table 2). Rat urinary metabolites from both control and pleurisy groups typically included the mammal and gut microbiota cometabolites25,31−33,39 such as hippurate, 3-indoxyl sulfate, and phenylacetylglycine (PAG), TCA cycle intermediates (citrate, 2ketoglutarate, succinate, and fumarate), choline metabolites,22,24 including dimethylglycine (DMG), trimethylamine (TMA), trimethylamine-oxide (TMAO), dimethylamine (DMA), and creatinine, together with some other metabolites such as taurine, acetate, allantoin, N-methylnicotinamide (NMNA), and N1methyl-4-pyridone-3-carboxamide (4-PY). Visual inspection revealed that in the acute phase of inflammation, rats with pleurisy had much higher plasma levels of lactate and OAG but much lower levels of lipids and creatine than controls. Urinary citrate and 2-ketoglutarate levels showed obvious differences and were much lower in pleurisy group than in controls in acute phase pleurisy (day 2). Multivariate data analysis was employed to reveal more details of metabolic changes associated with pleurisy development. Carrageenan-Induced Metabonomic Changes in Plasma

PCA results showed that three plasma samples from control group and one from the pleurisy group appeared to be outliers with excessively higher lactate level (data not shown). Therefore, these samples were excluded from subsequent analysis. In the resolution phase (at day 6 post-treatment), the results from the hierarchical cluster analysis (Figure S2 in the Supporting Information) confirmed the presence of these aforementioned subgroups (P6R and P6L). In the acute phase, good OPLS-DA model quality for control and pleurisy group was illustrated with R2X and Q2 values together with the CV-ANOVA, giving a p value of ∼0.002 (Figure 3A). Six days after carrageenan injection, OPLS-DA models also showed some metabonomic differences between subgroup P6R and controls (Figure 3B), although even smaller differences were found between subgroup P6L and controls. Significant differences were observable between two subgroups highlighted by higher levels for TG, lipids, and citrate

Carrageenan-Induced Fatty Acid Changes in Plasma

To obtain the detailed changes of plasma fatty acids resulting from the carrageenan-induced inflammation, we further analyzed the fatty acid composition of rat plasma samples from controls and pleurisy group using GC−FID/MS methods. The results showed remarkable changes of individual fatty acids associated with pleurisy development and progression (Table S1 in the Supporting Information). In the acute phase, pleurisy group had significantly lower levels in many fatty acids than controls (Figure 5528



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Information). In contrast, the levels for TCA cycle intermediates (citrate and 2-ketoglutarate) and acetate were higher in the P6L subgroup than in controls, whereas the levels for gut microbiotarelated metabolites (TMAO, PAG, and 3-indoxyl sulfate),24,25,39 taurine and NMNA were lower in P6L group (Table S2 in the Supporting Information). Dynamic urinary metabolite changes were clearly observable as a function of pleurisy development (Figure 5). TCA cycle intermediates responded vigorously

4A) including some SFAs (C16:0, C18:0, and C20:0), MUFAs (C18:1n7 and C20:1n9), and PUFAs (C18:2n6, C20:2n6,

Figure 4. Carrageenan-induced changes in plasma fatty acids against the levels in control rats. (A) Day 2 post-carrageenan treatment and (B) P6R subgroup at day 6 post-treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

C20:3n6, C20:4n6, C20:5n3, and C22:6n3). In the resolution phase (day 6 post-carrageenan treatment), two subgroups behaved differently in their fatty acids. Rats in the P6R subgroup had significantly higher levels for C16:0 and some UFAs (C18:1n7, C18:2n6, and C20:3n3) than controls (Figure 4B). However, P6L subgroup showed no differences from controls in terms of fatty acids (Table S1 in the Supporting Information). Carrageenan-Induced Metabonomic Changes in Rat Urine

OPLS-DA results (Figure S6 in the Supporting Information) showed pleurisy-associated dynamic changes for the rat urinary metabonomes. At day 2 post-treatment, carrageenan-injection caused significant level decreases for urinary citrate, 2ketoglurate, succinate, and DMG together with significant level elevations for creatinine and an unknown metabolite (Figure S6 in the Supporting Information). At day 4 post-treatment, significant metabonomic differences were also observable between the pleurisy and control groups (Figure S6 in the Supporting Information) highlighted with higher levels for allantoin and some aromatic metabolites. At day 6 posttreatment, two subgroups behaved differently. While the OPLS-DA model from controls and P6L subgroup showed good quality, such model for P6R subgroup and controls was not good enough to facilitate further analysis (Figure S6 in the Supporting Information). We further conducted the Student’s t tests for the metabolites with significant intergroup differences using the integrals of metabolite signals having little overlapping. The results indicated that at day 2 post-treatment, TCA intermediates (citrate, succinate, fumarate) were decreased compared with controls, whereas the choline metabolites (choline, phosphocholine, and creatinine) were elevated (Table S2 in the Supporting Information). Furthermore, the level of allantoin (the end product of purines) was increased. At day 4 post-treatment, gut microbiota-related metabolites (TMAO and PAG)24,25,39 and NMNA showed level declines compared with controls, whereas allantoin and 4-PY were elevated (Table S2 in the Supporting Information). At day 6 post-treatment, the carrageenan-induced changes for all urinary metabolites for rats in P6R subgroup showed no significant differences from those in controls, with exception of only taurine, whose level was lower in the P6R subgroup than in controls (Table S2 in the Supporting

Figure 5. Dynamic changes of urinary metabolites associated with progression of the carrageenan-induced pleurisy. At day 6 posttreatment, the empty symbols represented P6R subgroup, whereas the solid ones were for P6L.

during the inflammation development processes; urinary citrate and succinate levels decreased more than 20% in acute phase (Figure 5) and gradually recovered between day 2 and day 4 postcarrageenan injection. The levels of citrate and 2-ketoglutarate were ∼20% higher than controls in P6L at day 6 post-treatment. It is also interesting to note that the gut microbiota-related metabolites changed obviously during the resolution phase with PAG decreased about 10−20% from day 2 to day 4 post-treated. Compared with controls, the metabolite of vitamin B3, NMNA, decreased for ∼25% from day 2 to day 4 post-treatment and to ∼40% for P6R subgroups at day 6 post-treated (Figure 5).

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DISCUSSION The development of inflammatory processes often undergoes an acute and a resolution phase. Many previous results have indicated that metabolic changes are accompanied by such processes, although the details remain to be explored. This study was aimed to investigate the dynamic metabolic changes associated with the development of pleurisy induced by carrageenan-injection, which was a well-established self-limiting local inflammation model. NMR-based metabonomics approach revealed metabonomic changes in rat plasma and urine with GC−FID/MS data for plasma fatty acids and clinical chemistry data as complementary information. The results clearly have 5529



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illustrated that the carrageenan-induced pleurisy is a self-limiting processes with two clear (acute and resolution) phases. Such local inflammation process in fact caused system metabonomic changes involving multiple metabolic pathways including fatty acid metabolism, protein biosynthesis, glycolysis, TCA cycle, mammal-gut microbial cometabolisms, catabolism of vitamin B3, choline, and purines. At day 6 post-treatment, the metabolic changes induced by carrageenan-injection were not completely resolved, and two subgroups (P6R and P6L) were clearly visible from both the phenotypic and metabonomic data, indicating the heterogeneity of the inflammation resolution.

and C20:3n3 were about 15−30% higher than those in controls (Figure 4B). In contrast, rats in the P6L subgroup showed no significant differences from corresponding controls in the levels of all plasma fatty acids. However, P6L rats had significantly higher plasma levels for tyrosine and NAG but lower levels for citrate, glutamine, and lactate than controls, part of which were reflected in the acute phase of pleurisy (e.g., NAG and albumin) (Table 1 and Figure S4 in the Supporting Information). This implies that the self-limiting nature for this pleurisy is reflected in the metabolic aspects, although fatty acid metabolism reprogramming is still ongoing for some animals 6 days after pleurisy induction. The duration required for the complete metabolic recovery remains to be studied together with the detailed metabolic regulations.

Lipid Metabolism

The carrageenan injection caused drastic changes in fatty acid metabolism highlighted by significant level reductions for rat plasma fatty acids (Figure 4) in the acute phase of pleurisy (at day 2 post-treatment). Many of the plasma fatty acids showed about 20−40% decreases compared with controls (Figure 4). This is in broad agreement with the previous findings of the significant decreases in total PUFAs upon carrageenan injection.49 We observed that both n3 and n6 PUFAs showed significantly decreases here, although the n6-to-n3 ratio actually increased significantly (Table S1 in the Supporting Information). This is consistent with what has been reported for the pro-inflammation effects of diets having high n6-to-n3 ratio.50,51 Since these two classes of fatty acids are both essential fatty acids, their ratio changes in blood plasma are most likely from the inflammationinduced fatty acid changes with constant dietary intakes. It is now well known that n3 PUFAs cannot be produced by mammals via de novo biosynthesis and have to be obtained through dietary sources mostly in the form of α-linolenic acid (ALA). ALA is then converted into eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) through a series of elongations and desaturations. Previous results indicated that EPA and DHA played some roles in anti-inflammation processes through multiple mechanisms involving the cell membrane, cytosol, and nucleus.52 A recent study on cellular systems reported that these n3 PUFAs caused dramatic changes in TLR4 and purinergic eicosanoid signaling, which was shunt for lipoxygenase pathway of the arachidonic acid metabolism, exerting anti-inflammatory effects.53 Although it was generally assumed that n6 PUFAs, especially arachidonic acid and its derived eicosanoids, were proinflammatory, this was probably an oversimplification because the downstream metabolites of n6 fatty acids changed with different stimulus and tissues.54 However, the concurrent decrease in plasma acetoacetate observed here further suggests that translocation of fatty acids from circulation into certain organs might also be an important response to the acute inflammation because catabolism of fatty acids via β-oxidation would have to elevate the levels of ketone bodies such as 3hydroxybutyrate, acetone, and acetoacetate. In the resolution phase (day 6 after carrageenan injection), the phenotypes of the pleurisy animals appeared to be heterogeneous with both thymus and spleen tissue weights together with pleural exudate volumes showing two subgroups, a better resolved group (P6R) and a less resolved one (P6L). The larger standard deviation for C16:0 in the pleurisy group than in controls also indicated the presence of heterogeneity. In fact, OPLS-DA results showed that the P6R subgroup had significantly higher levels in fatty acids and triglycerides than controls (Figure 3B), which agreed well with the clinical chemistry data (Table 1). It is interesting to observe that in this subgroup, the plasma levels for C16:0, C18:1n7, C18:2n6,

TCA Cycle and Glycolysis

Significant elevation of plasma succinate in the acute phase of pleurisy suggested that TCA cycle was enhanced with the development of inflammation, although alterations of succinate dehydrogenase were also a possibility.56,57 Because no obvious changes of plasma glucose were observed in both NMR and clinical chemistry data (Table 1), such elevation of succinate was probably from enhanced glycolysis at the inflammatory sites. Significant elevation of lactate in plasma was probably also a consequence of such enhanced glycolysis because elevation of lactate in the local inflammatory tissues via glycolysis was one of the most common acute phase reactions.55 This agreed well with the inflammation-induced elevation of lactate dehydrogenase in the carrageenan-caused pleural fluids.56 In fact, some previous studies on both rats and human patients suggested that the large quantity of lactate from the inflammation sites was more likely from the glycolytic metabolism of inflammation cells rather than a marker for tissue hypoxia.57 Further evidence seemed to indicate that such hyperlactataemia following inflammation was probably a result of the adrenaline-stimulated aerobic glycolysis.57 This is understandable because inflammatory cells, especially leukocytes can actively produce lactate as the predominant end product of glycolysis, with macrophages regenerating their energy stores during phagocytosis.58 Increased levels of lactate in synovial fluid have also been well-documented in various types of inflammation.58 Six days post carrageenaninjection, two subgroups were observed in the pleurisy group, with P6R rats (better resolved subgroup) showing no significant differences from controls for all TCA cycle intermediates and lactate (Table S2 in the Supporting Information). The less resolved subgroup (P6L) showed lower levels for citrate and lactate than controls (Table S2 in the Supporting Information). This indicates that acute phase responses of the TCA cycle and glycolysis have come to a resolve to some degree around a week after inflammation, which is consistent with the previous observations of disappearance of inflammation cells at the injury sites at the end of resolution phase.57,58 Significant level declines for urinary citrate, succinate, and fumarate in pleurisy group at day 2 post-treatment further confirmed the disruptions of TCA cycle caused by acute inflammation. Metabolisms of Proteins and Amino acids

Our observation of the carrageenan-induced significant elevations for OAG and NAG in blood plasma is a typical acute phase response (APR) with these proteins known as “acute phase” glycoproteins.59 Hypoalbuminaemia is normally considered as a measure of malnutrition or inflammation.60 However, we observed no significant differences in body weights for the control and pleurisy rats, indicating no significant differences in 5530



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microbiota functions respond to inflammation72,73 much more in the resolution phases. Choline metabolites in urine showed significant changes at the acute phase of pleurisy (day 2 postinduction) with elevations of choline, phosphocholine, and creatinine but level decline for DMG, showing promoted excretion of choline in the forms of itself and creatinine. It remained unknown about the driving force for this event, although osmotic regulations were likely involved because taurine, a well-known osmolyte, also showed declines during the resolution phase of pleurisy (at day 6 postinduction). Consistent level decline for urinary NMNA (a metabolite of nicotinamide) was obviously observable at day 4 and day 6 (for the less resolved subgroup) post-carraggenan induction. This together with the elevation of N1-methyl-4-pyridone-3-carboxamide (4-PY) at day 4 suggested that catabolism of nicotinamide (vitamin B3) was involved in the progression of inflammation. It is common knowledge now that nicotinamide is a component of NAD participating in intracellular respiration to oxidize fuel substrates. This vitamin B3 is readily metabolized into N1methylnicotinamide in mammalian liver, which can be further oxidized to produce 4-PY.74,75 Enhanced oxidative stress is also implied because 4-PY is an oxidization metabolite of NMNA. The elevation of urinary allantoin at day 2 and day 4 postinduction supports this notion because allantoin is the final metabolite of oxidative catabolism for purines.

food intake. Therefore, the carrageenan-induced significant level decreases for serum albumin (Table 1) observed here are probably due to albumin leakage61 into large quantity of pleural exudate (about 2−8 mL/rat) rapidly produced upon carrageenan injection, which is also a typical APR. The elevation of serum globulin observed in the pleurisy model here is in good agreement with what has been reported previously for the carrageenan-induced elevations for globulins.49 Because there were no obvious liver damages observed, this hyperglobulinemia is probably a secondary cause of hypoalbuminemia acting as a compensatory mechanism to maintain plasma osmolarity.62 This point is further reinforced by the significant decrease in the albumin-to-globulin ratio (Table 1), which has already been reported in previous studies.62 The decreases in rat plasma HDL with the concurrent elevation of LDL in the acute phase of inflammation (Table 1) agreed with the observations in the LPS-induced inflammation63,64 showing changes of apolipoprotein composition in HDL.65 Although it was considered to be anti-inflammatory in the basal state,66 HDL could be altered in its structure during acute inflammation and became pro-inflammatory by reducing the levels of aryl hydrolase and paraoxonase.64 It is now known that with acute-inflammation HDL undergoes a major change in apolipoprotein composition and becomes so-called acute-phase HDL (AP-HDL).67 The carrageenan-induced reduction of plasma cholesterol (Table 2) is consistent with previous observations of the transiently reduced cholesterol efflux to plasma in patients under an acute inflammation.68 This is also supportive of the notion that inflammation effects on HDL metabolism probably accelerated HDL catabolism, leading to substantial remodeling of the HDL particles. It is interesting to point out that this dysfunctional HDL is also well-documented for its associations with diabetes and coronary artery diseases,69 which are all related to chronic inflammation. The plasma levels of some amino acids were elevated including lysine, proline, histidine, and phenylalanine during the acute phase of inflammation. This agreed well with previous findings of the infection-related elevations in serum phenylalanine and phenylalanine−tyrosine ratio probably due to increased catabolism connective tissues.70 This notion is further supported by the elevation of serum proline observed in animal models of inflammatory bowel disease and arthritis.71 Six days after carrageenan-injection (i.e., in the resolution phase), serum albumin level remained significantly lower in pleurisy group than in controls. This also indicates that 6 days were not long enough to enable inflammation to be completely resolved, as shown by metabolic data. The resolution phase of inflammation was heterogeneous with the presence of two subgroups, showing different reprogramming for the acute phase proteins and some amino acids.

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CONCLUSIONS This study showed that systems metabolic changes were closely associated with the onset and progression of the carrageenaninduced pleurisy in rats with acute and resolution phases. In the acute phase, a large quantity of pleurisy exudate (2−8 mL) was produced, accompanied by significant declines of serum albumin but elevation of globulin levels. Alterations in energy, lipoproteins, and protein metabolisms were the major responses to acute inflammatory processes. Such responses included substantial dysfunction of HDL, drastic reduction of serum fatty acids, elevation of acute phase proteins (NAG and OAG) and some amino acids, enhanced glycolysis, and disruptions of energy metabolism involving TCA cycle. Some of these changes were still observable 6 days after treatment including serum albumin and acute phase proteins together with the reprogramming of lipid metabolism, glycolysis, and TCA cycle. The progression of pleurisy also affected vitamin B3 and choline metabolisms, gut microbiota functions accompanied by oxidative stress to some extents. The results further suggested that 6 days were not sufficient to complete inflammation resolution. These findings provided essential metabolic information associated with the inflammation developments and demonstrated the combined NMR and GC−FID/MS analysis of metabolisms as a powerful approach for understanding the biochemical aspects of inflammation. Further studies are needed to find the metabonomic responses of specific organs toward such local inflammation at a specific site and to understand the generic and specific responses of mammals toward different inflammatory processes.

Gut Microbiota and Other Metabolisms

The progression of pleurisy was accompanied by level changes for some gut microbiota-related metabolites in urine, especially during the resolution phases. At day 4 after carrageenantreatment, PAG and TMA levels were significantly lower in pleurisy rat urine than in control ones. At day 6 post-treatment, two subgroups behaved differently as compared with controls. Rats in the P6L (less resolved) subgroup showed significantly lower urinary levels for PAG, 3-indoxyl sulfate, and TMAO (a choline metabolite from gut micriobiota24) than controls, whereas those in the P6R (better resolved) subgroup showed no significant differences from controls. This implies that the gut

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ASSOCIATED CONTENT

S Supporting Information *

Fatty acids in plasma of rats. Significantly changed rat urinary metabolites induced by carrageenan-injection. Phenotypic characteristics for rats treated with carrageenan-injection and 5531



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(13) Heinrich, P. C.; Castelltt, J. V.; Andust, T. Interleukin-6 and the acute phase response. J. Biochem. 1990, 265, 621−636. (14) Vane, J.; MIitchell, J.; Appleton, I.; Tomlinson, A.; Bailey, D.; Croxtall, J.; Willoughby, D. Inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation. Proc. Natl. Acad. Sci. 1994, 91, 2046−2050. (15) Smith, W. L.; Dewitt, D. L.; Garavito, R. M. Cyclooxygenases: structural, cellular, and molecular biology. Annu. Rev. Biochem. 2000, 69, 145−182. (16) Simon, L. S. Role and regulation of Cyclooxygenase-2 during Inflammation. Am. J. Med. 1999, 106, 37−42. (17) Salvemini, D.; Wang, Z. Q.; Wyatt, P. S.; Bourdon, D.; Marino, M. H.; Manning, P. T.; Currie, M. G. Nitricoxide: a key mediator in the early and late phase of carrageenan-induced rat paw inflammation. Br. J. Pharmacol. 1996, 118, 829−838. (18) Nicholson, J. K.; Lindon, J. C.; Holmes, E. ‘Metabonomics’: understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica 1999, 29 (11), 1181− 1189. (19) Tang, H. R.; Wang, Y. L. Metabonomics: a revolution in progress. Prog. Biophys. Biochem 2006, 33 (5), 401−417. (20) Wang, Y.; Tang, H.; Holmes, E.; Lindon, J. C.; Turini, M. E.; Sprenger, N.; Bergonzelli, G.; Fay, L. B.; Kochhar, S.; Nicholson, J. K. Biochemical characterization of rat intestine development using highresolution magic-angle-spinning 1H NMR spectroscopy and multivariate data analysis. J. Proteome Res. 2005, 4 (4), 1324−1329. (21) Wang, Y.; Holmes, E.; Comelli, E. M.; Fotopoulos, G.; Dorta, G.; Tang, H.; Rantalainen, M. J.; Lindon, J. C.; Corthésy-Theulaz, I. E.; Fay, L. B. Topographical variation in metabolic signatures of human gastrointestinal biopsies revealed by high-resolution magic-angle spinning 1H NMR spectroscopy. J. Proteome Res. 2007, 6 (10), 3944− 3951. (22) Tian, Y.; Zhang, L.; Wang, Y.; Tang, H. Age-related topographical metabolic signatures for the rat gastrointestinal contents. J. Proteome Res. 2011, 11 (2), 1397−1411. (23) Wang, Y.; Cloarec, O.; Tang, H. R.; Lindon, J. C.; Holmes, E.; Kochhar, S.; Nicholson, J. K. Magic angle spinning NMR and 1H-31P heteronuclear statistical total correlation spectroscopy of intact human gut biopsies. Anal. Chem. 2008, 80 (4), 1058−1066. (24) Dumas, M.-E.; Barton, R. H.; Toye, A.; Cloarec, O.; Blancher, C.; Rothwell, A.; Fearnside, J.; Tatoud, R.; Blanc, V.; Lindon, J. C. Metabolic profiling reveals a contribution of gut microbiota to fatty liver phenotype in insulin-resistant mice. Proc. Natl. Acad. Sci. 2006, 103 (33), 12511− 12516. (25) Xu, W.; Wu, J.; An, Y.; Xiao, C.; Hao, F.; Liu, H.; Wang, Y.; Tang, H. R. Streptozotocin-induced dynamic metabonomic changes in rat biofluids. J. Proteome Res. 2012, 11 (6), 3423−3435. (26) Zhang, X.; Wang, Y.; Hao, F.; Zhou, X.; Han, X.; Tang, H. R.; Ji, L. Human serum metabonomic analysis reveals progression axes for glucose intolerance and insulin resistance statuses. J. Proteome Res. 2009, 8 (11), 5188−5195. (27) He, Q.; Ren, P.; Kong, X.; Wu, Y.; Wu, G.; Li, P.; Hao, F.; Tang, H. R.; Blachier, F.; Yin, Y. Comparison of serum metabolite compositions between obese and lean growing pigs using an NMR-based metabonomic approach. J. Nutr. Biochem. 2012, 23 (2), 133−139. (28) Brindle, J. T.; Antti, H.; Holmes, E.; Tranter, G.; Nicholson, J. K.; Bethell, H. W.; Clarke, S.; Schofield, P. M.; McKilligin, E.; Mosedale, D. E. Rapid and noninvasive diagnosis of the presence and severity of coronary heart disease using 1H-NMR-based metabonomics. Nat. Med. 2002, 8 (12), 1439−1445. (29) Marchesi, J. R.; Holmes, E.; Khan, F.; Kochhar, S.; Scanlan, P.; Shanahan, F.; Wilson, I. D.; Wang, Y. Rapid and noninvasive metabonomic characterization of inflammatory bowel disease. J.Proteome Res. 2007, 6 (2), 546−551. (30) Bjerrum, J. T.; Nielsen, O. H.; Hao, F.; Tang, H.; Nicholson, J. K.; Wang, Y.; Olsen, J. Metabonomics in ulcerative colitis: diagnostics, biomarker identification, and insight into the pathophysiology. J.Proteome Res. 2009, 9 (2), 954−962.

controls. Hierarchical cluster analysis of plasma data for rats under the carrageenan-injection. OPLS-DA results showed significant plasma metabonomic differences between two subgroups in the resolution phase of pleurisy. Results from the Student’s t test indicated that rats in the P6L subgroup had significantly different metabonomes from controls. Results from the Student’s t test indicated that rats in the pleurisy group had significantly lower plasma levels for PUFAs, UFAs, and ToFAs but higher levels for acetyl-glycoproteins than controls. OPLSDA results for rat urinary metabonomic changes induced by carrageenan-injection. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-(0)27-87198430. Fax: +86-(0)27-87199291. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the financial support from the Ministry of Science and Technology of China (2009CB118804, 2010CB912501, and 2012CB934004), National Natural Science Foundation of China (20825520, 21175149, and 21221064), and Chinese Academy of Sciences (KJCX2-YW-W13 and KSCX1YW-02).

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