Experimental Lung Research, 40, 460–466, 2014 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 0190-2148 print / 1521-0499 online DOI: 10.3109/01902148.2014.947008

ORIGINAL ARTICLE

Metabolomic analysis of lung epithelial secretions in rats: An investigation of bronchoalveolar lavage fluid by GC-MS and FT-IR

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Wajhul Qamar, Syed Rizwan Ahamad, Raisuddin Ali, Mohammad Rashid Khan, and Abdul Rahman Al-Ghadeer Central Laboratory, Research Center, College of pharmacy, King Saud University, Riyadh, Saudi Arabia AB STRACT Objective: Rat bronchoalveolar lavage fluid (BALF) metabolome can be used to obtain valuable, precise, and accurate information about underlying lung conditions in an experiment. The present study focuses on the evaluation of the lung epithelium metabolome in a rat model using techniques including bronchoalveolar lavage, gas chromatography-mass spectroscopy (GC-MS), and Fourier transform infrared spectroscopy (FT-IR). Materials and methods: Untargeted metabolites in BALF were extracted in ethyl acetate and derivatized by standard methods for the analysis by GC-MS. FT-IR spectra of ethyl acetate extract of BALF were obtained and read for the characteristic fingerprint of rats under investigation. Analyses were done in individual animals to obtain consistent data. BALF cells were counted by flow cytometry to monitor any inflammatory condition in rats. Results: FT-IR analysis finds two peaks which are characteristically different from the extract medium, which is ethyl acetate. FT-IR peaks correspond to that of amino acids and carbohydrates, including β-D-glucose, α-D-glucose, and β-D-galactose. GC-MS evaluation of the BALF finds several products of the metabolism or its participants. Main compounds in the BALF detected by GC-MS include succinate, fumarate, glycine, alanine, 2-methyl-3oxovaleric acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octanoic acid, trans-9-octadecanoic acid, octadecanoic acid, and Prostaglandin F1α. Conclusion: Several research reports reveal metabolomic parameters in murine model lung tissue or BALF, but they rarely reported a complete metabolomics model profile, particularly in rats. The present data of GC-MS and FT-IR suggest that the set up can be exploited to study metabolomic alterations in several lung conditions including acute lung toxicity, inflammation, asthma, bronchitis, fibrosis, and emphysema. KEYWORDS bronchoalveolar lavage fluid (BALF), FT-IR fingerprint, GC-MS, lung, metabolomics

In murine models, BALF is applied for a wide range of experiments associated with the lung; however, the method is not feasible in humans and needs clinicians with specialist training. BALF has been used for a number of analyses such as lung inflammation and associated alterations in biochemical composition. Recent advances in the field of analytical sciences have opened new arenas for researchers. The past decade has seen the development of various revolutionary methodologies in the research field. Analytical methods are expanding their area of application not only to chemistry but also to biological sciences. Instruments like gas chromatography-mass spectroscopy (GC-MS), liquid chromatography-mass spectroscopy (LC-MS), nuclear magnetic resonance

INTRODUCTION Bronchoalveolar lavage fluid (BALF) serves as a biological sample obtained from the lungs, and it directly represents the excretion of the pulmonary surfactant.

Received 10 June 2014; accepted 17 July 2014 The authors thank the Research Center, College of Pharmacy, King Saud University, Riyadh, Kingdom of Saudi Arabia. Address correspondence to Wajhul Qamar, Central Laboratory, Research Center, College of pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia. E-mail: [email protected] and qamarjh@rediffmail. com

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BALF Metabolomics by GC-MS and FT-IR

(NMR), etc., have been proved to analyze several biological phenomena in animals as well as human subjects [1–3]. Their use in clinical pharmacology is very much accepted by the scientific community as they can provide the much desired information about “drug–biological system interaction.” Metabolomics is the field of clinical research where the end products of the metabolism (metabolic profile) of an individual are monitored by means of analytical methods like GC-MS, LC-MS, and NMR, which can be applied to BALF as well. The area is emerging and promising in fields including clinical diagnosis, clinical research, pharmacology of drugs under development, and toxicological studies [4, 5]. Metabolic intermediates, which are mainly low molecular weight compounds (LMCs), reflect the actions of proteins (enzymes) in biochemical reactions that are metabolic pathways, and, thus, represent biological states in a way analogous to proteomes. In line with transcriptomic and proteomic responses to environment and stress, the metabolome is also responsive to various kinds of toxicant exposures and diseased conditions. In case of BALF, it can reflect metabolomic alterations that indicate direct interactions with the environment or any underlying disease/disorder. A large number of drugs have been evaluated by means of metabolomic approaches in various animal and human studies. Metabolomic approaches can be applied to study the metabolic changes associated with pathological conditions [6]. In case of studies on pharmaceuticals, metabolomic evaluations can provide detailed information regarding the effectiveness of the drug as well as the probable toxic events in the lungs. These methods are well accepted and appreciated in the scientific community and considered promising in the area of pharmacy for drug discovery and development [7–9]. MS-based analyses offer an important alternative approach to metabolomics. The greatest potential advantage of MS-based methods is their sensitivity. MS analyses can detect molecules at levels up to 10,000-fold lower than NMR [10, 11]. Present manuscript focuses on the analysis of BALF from rats by GC-MS in order to obtain an MS-based profile. Ellis and Goodacre [12] discussed Fourier transform infrared spectroscopy (FT-IR) applications in metabolomics. FT-IR can provide qualitative data, based on functional groups in the compounds, in the form of a spectral fingerprint of the biological sample under investigation. This fingerprint may serve as a characteristic variable for a particular biological sample. We adopted FT-IR as supportive tool along with GC-MS for metabolomic analysis of rat BALF.

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MATERIALS AND METHODS Chemicals Ethyl acetate, hexane, pyridine, and methoxamine were purchased from Merck, Germany. All other chemicals used were of highest purity grade.

Animals Pathogen-free healthy male rats of Wistar strain were used in this study. Animals were obtained from the Animal Care Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. The rats were approximately 12 weeks old (weight, 200–250 g). Ten animals were housed in two different polypropylene cages, five rats per cage, and were kept in a room maintained at 25 ± 2◦ C with a 12-hour light/dark cycle. Animals were given free access to standard laboratory animal feed and water ad libitum.

Bronchoalveolar Lavage Bronchoalveolar lavage fluid (BALF) was collected on the basis of methods described elsewhere [13, 14], with some modifications adopted in our previous studies [15, 16]. The trachea of anesthetized animals was exposed; a cannula was inserted into the trachea and was ligated using a thread. Chilled phosphatebuffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4) at a volume of 25 mL/kg body weight was injected into the lungs via a syringe fitted with a 20-gauge tracheal cannula. The PBS was allowed to remain in the lungs for 30 seconds, then retrieved and re-instilled with the help of syringe. This was repeated thrice with the same solution. The percentage yield collected was 78.76 ± 4, and the collected volumes of BALF from different animals were not significantly different. The BALF thus obtained was centrifuged (300g, 10 minutes) to collect the cell pellet for cell analysis/count. The acellular supernatant was divided into aliquots and stored at −20◦ C. Animal remains were submitted to the institutional animal house facility for their disposal according to international guidelines.

Cell Count in BALF The present study was focused on obtaining the BALF metabolome of healthy rats, and any kind of acute or chronic lung condition in the experimental animals might affect the results. Cells in BALF were counted as an initial check for any underlying

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inflammatory process in the lungs of the rats. The cell pellet obtained after centrifugation of BALF was washed three times with PBS. This washing was performed immediately after BALF collection. Thereafter, the obtained cell pellet was suspended in PBS and the cells were counted using flow cytometry (Beckman Coulter, FC500) using forward scatter and side scatter, and the flow rate of the machine was set on 60 μL/s (medium rate). A total of 5000 cells were counted in a single run of the sample.

detection was carried out in electron ionization mode by scanning at 40-600 (m/z). Finally, unknown compounds were identified by comparing the spectra with that of the National Institute of Standard and Technology 2005 Library and Wiley AccessPak v7 May, 2003 library. The total time required for analyzing a single sample was 58 minutes. A blank was run after every five samples.

BALF Processing for GC-MS and FTIR Evaluations

The FT-IR spectra of the ethyl acetate extract of rat BALF were obtained using Perkin Elmer, Spectrum BX FT-IR system. Briefly, clean and dry sodium chloride (NaCl) plates were taken and one drop of BALF extract was placed on one of the plates using a micropipette; then, a second plate was placed on the sample-containing plate in a way that no air bubbles were trapped. This formed a thin layer of extract between the two plates. These plates were then mounted in a liquid cell holder and the absorbance was measured. Spectral fingerprint was obtained and compared with spectrum of ethyl acetate. The BALF samples were analyzed on the basis of absorption peaks which were different from that of the ethyl acetate and also on the basis of the potential library matches in the instrument.

To extract the constituents/metabolites from the BALF we adopted the method reported by Fabiano et al. [17] in which ethyl acetate was used as the extraction medium. The method was slightly modified according to our experimental requirements and standardization. Briefly, in 500 μL BALF, 400 μL ethyl acetate was added and the mixture was vigorously vortexed for 5 minutes. The mixture was centrifuged for 10 minutes, 15,000 rpm (21,000g) at 4◦ C, and the supernatant was collected for analysis by GC-MS and FT-IR.

GC-MS Analysis For GC-MS analysis, a 300-μL sample of supernatant was placed in a GC vial and evaporated under nitrogen stream. Into this vial, 100 μL of methoxamine/pyridine (20 mg/mL) was added. After overnight incubation at room temperature, 100 μL N, O-bis-trimethyl tri-fluoroacetamide (BSTFA) was added along with 40 μL of hexane containing 5 μL undecane as the internal standard (Fabiano et al., 2011). This final mixture was subjected to analysis by GC-MS. The GC-MS analysis was accomplished in a gas chromatograph (Perkin-Elmer, Autosystem XL) linked to a mass spectrometer (Turbomass) available at the Research Center, College of Pharmacy King Saud University. An aliquot of 2 μL of extract was injected into the Elite-5MS column of 30 m × 0.25 mm internal diameter of 0.25-μm film thickness glass capillary column using the following temperature program: initial oven temperature of 30◦ C for 2 minutes, increasing to 300◦ C at a rate of 5◦ C/min with a hold for 2 minutes at the ending temperature. The injector temperature was maintained at 250◦ C. The interface temperature was 200◦ C and the GC line temperature is at 220◦ C; the electron energy used was 70 eV. The scan duration was 1.2 second and the inter scan delay was 0.2 seconds. Helium was used as a mobile phase at a flow rate of 1.0 mL/min. Mass spectral

Fourier Transform Infrared Spectroscopy (FT-IR) Analysis

Statistical Analysis Data obtained from different animals (GC-MS, BALF cell count and BALF volume) were statistically analyzed for normal distribution by the Kolmogorov–Smirnov test using GraphPad Instat software.

RESULTS BALF Cell count to Check for any Interfering Inflammation Total cell count from different BALF samples was found to be 0.051 ± 0.007 million cells/mL BALF. Statistical analysis found the number to be normally distributed among animals. The results represent a healthy, non-inflammatory lung. To confirm this further, the T-lymphocyte population infiltration was also analyzed using CD3 antibodies and results were found to be in the normal range (data not given).

Metabolites Detected by GC-MS A total of 33 detectable peaks were selected for metabolite identification in the National Institute Experimental Lung Research

BALF Metabolomics by GC-MS and FT-IR TABLE 1.

Metabolites Detected in BALF of Rats by GC-MS

Metabolite

Pathway

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Succinic acid Fumaric acid Glycine Alanine 2-methyl-3-oxovaleric acid Dodecanoic acid Tetradecanoic acid Hexadecanoic acid Octanoic acid Trans-9-octadecanoic acid Octadecanoic acid Prostaglandin F1α

Krebs (TCA) cycle Krebs (TCA) cycle Amino acid Amino acid, glycolysis Amino acid Fatty acid Fatty acid Fatty acid Fatty acid Fatty acid Fatty acid Arachidonic acid/cyclooxygenase

of Standard and Technology (NIST) 2005 Library and Wiley AccessPak v7 May, 2003 library. Out of these, 12 were found to have metabolomic significance (Table 1). Figure 1 shows the chromatogram obtained by the GC-MS analysis of the derivatized BALF. The metabolites detected include two from the Krebs cycle (succinic acid and fumaric acid), three from amino acid metabolism (2-methyl3-oxovaleric acid, glycine, and alanine), six from fatty acid/lipid metabolism (dodecanoic acid, tetrade-

canoic acid, hexadecanoic acid, octanoic acid, trans9-octadecanoic acid, and octadecanoic acid), and one from arachidonic acid pathway (Prostaglandin F1α).

FT-IR Fingerprint of the BALF Spectral fingerprints obtained from FT-IR show two new absorption peaks in BALF samples when compared with the spectrum of the extraction medium, which is ethyl acetate (Figure 2a and Figure b). Peaks A and B, which were detected around 3460 cm−1 and 1650 cm−1 respectively, are unique to the ethyl acetate medium, which was used to extract metabolites from the different samples of the BALF from healthy rats. Peak analysis reveals that these two regions may represent amino acids and sugars. However, GC-MS could confirm the presence of amino acids only in the very same samples.

DISCUSSION Biochemical composition of samples varies between healthy and diseased/treated animals and provides

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FIGURE 1. Different peaks, representing different compounds, obtained from GC-MS analysis of rat BALF. Of these, only 12 compounds were found to be have metabolomic significance (see Table 1). Numerical values of the peaks are “retention time” of individual compounds.  C

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This overlay figure compares the spectral fingerprint of ethyl acetate extract of BALF from different animal pools (BALF EA1 to 5) with spectral fingerprint of ethyl acetate. Column A and B highlight the absorption peaks which are different from ethyl acetate peaks and are characteristic of healthy rat BALF. The A is around 3460 cm−1 and the B is around 1650 cm−1 .

FIGURE 2a.

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This is a spectral fingerprint of BALF obtained from healthy rats. Peaks A and B are unique to that of extraction medium, ethyl acetate. The A is around 3460 cm−1 , and the B is around 1650 cm−1 . Analysis of spectra reveals that the range of these peaks is characteristic of the presence of amino acids.

FIGURE 2b.

Experimental Lung Research

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BALF Metabolomics by GC-MS and FT-IR

the basis for metabolomic analysis, which has gained credence among the biomedical and pharmaceutical research community. Lungs are susceptible to injuries due to direct interaction with the environment, large surface area, high vasculature, and very thin epithelium. Any diseased condition may impose a misleading interference in the data that is sought to be obtained as a reference from healthy animals. Inflammation is the most common among various lung conditions including asthma, bronchitis, emphysema, fibrosis, etc., and may alter the biochemical composition within and affect the metabolome of the lungs. In the present experiment, results from the BALF cell count of different animals is found to be in agreement with previously reported observations in control/untreated healthy animals [15, 18]. BALF cell count is a reliable and commonly adopted method to observe inflammation in the lung [19]. Rat models have been used to study various lung ailments [20, 21]. BALF metabolomic information from healthy rats may serve as a tool to design an experiment to investigate an abnormal lung condition in a preclinical set up which may draw the interest of researchers from pharmacological and toxicological fields of study. In the present investigation, we focused on obtaining the BALF metabolomic profile from healthy rats. GC-MS analysis of the BALF samples revealed 12 compounds of interest from different metabolic pathways. Prostaglandin F1α is one of the metabolites detected in rat BALF by GC-MS (Table 1). It is a metabolite of prostacyclin (prostaglandin I2 or PGI2). PGI2 is negatively associated with pulmonary arterial hypertension, and the pathway is one of the targets for therapeutic interventions [22]. In addition, pulmonary hypertension has been associated with lung diseases like chronic obstructive pulmonary disease (COPD) and interstitial lung disease along with other heart and blood disorders [23]. The level of prostaglandin F1α in BALF may have significant role in determining the linkage between inflammationassociated pulmonary hypertension. Intermediate metabolites of the Krebs cycle (citric acid cycle) were also detected by GC-MS. These include four carbon intermediates, succinic acid, and fumaric acid, which are part of the energy-generating pathway. Altered levels of these metabolites can reflect interference in the pathway and health condition. Other metabolites detected in the BALF provide information regarding two pathways; (i) amino acid metabolism pathway: metabolites include glycine, alanine, and 2-methyl-3-oxovaleric acid (a metabolite of isoleucine). In addition, alanine participates in the glycolysis pathway; (ii) fatty acid metabolism pathway: metabolites include dodecanoic acid, tetrade C

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canoic acid, hexadecanoic acid, octanoic acid, trans9-octadecanoic acid, and octadecanoic acid. Fatty acids play a crucial role in energy metabolism in addition to the synthesis of phospholipid bilayer and several hormones. In case of the lung, they play an essential role in the synthesis of surfactants that protect lung alveoli from collapse. Dipalmitoylphosphatidylcholine is one of the major lung surfactants, synthesized by combining hexadecanoic acid (palmitic acid) with phosphatidylcholine. In this regard, detection of hexadecanoic acid in rat BALF along with other fatty acids can be presented as a crucial piece of information in order to explain the metabolic signature of diseased lung. A recent report by Ho et al., [24] revealed alterations in lipid metabolites in BALF of the murine model of house dust-mite-induced allergic asthma. Moreover, alterations in energy metabolism pathways were reported in the same study. FT-IR has been applied to monitor several biological samples, from isolated cells to whole organisms [25]. The spectral fingerprint obtained in the present investigation may be suggested as characteristic of normal, healthy rat lungs when following the reported protocol. However, FT-IR alone did not reveal much of the metabolomic composition of the BALF, but it surely played a supportive role in understanding the results obtained from GC-MS analysis. FT-IR may be applied as a supportive tool in metabolomics studies along with other techniques such as LC-MS and NMR. By this, it can be concluded that metabolic fingerprinting by FT-IR and specific identification of metabolites by GC-MS mutually support each other in the analysis of the metabolomic profile of BALF. Lung epithelial secretions are essential for the respiratory process, which includes gaseous exchange, and protection of lung epithelium from inhalant pathogens and xenobiotics. BALF directly represent the lung epithelial secretions, however diluted, and serves as a biological specimen for biomedical investigations including metabolomics [26, 27]. Moreover, metabolomics has gained a place in various preclinical studies, and can serve as a reliable tool for toxicological and pharmacological investigations. The number of the metabolites detected in rat BALF remained very limited as compared with the number in serum, as reported previously. Several other studies have also reported lesser numbers of metabolites in BALF [17, 27]. This can be attributed to the fact that the lavage medium is aqueous and the lung epithelial secretions contain less biochemical products compared with the serum. However, with our observation of the present data and extraction of BALF with other solvents such as methanol (data not given), it can be suggested that the extraction medium, for the purpose of metabolomic studies with BALF, needs to

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be explored further. It is also suggested that the FTIR plays a supportive role in the analysis of BALF metabolome and it may be applied to the analysis in other metabolomic studies including serum, cerebrospinal fluid, urine, saliva, etc. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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[14] Reynolds HY: Bronchoalveolar lavage. Am Rev Respir Dis. 1987;135:250–263. [15] Qamar W, Sultana S: Farnesol ameliorates massive inflammation, oxidative stress and lung injury induced by intratracheal instillation of cigarette smoke extract in rats: an initial step in lung chemoprevention. Chem Biol Interact. 2008;176: 79–87. [16] Qamar W, Khan AQ, Khan R, Lateef A, Tahir M, Rehman MU, Ali F, Sultana S: Benzo(a)pyrene-induced pulmonary inflammation, edema, surfactant dysfunction, and injuries in rats: alleviation by farnesol. Exp Lung Res. 2012;38:19–27. [17] Fabiano A, Gazzolo D, Zimmermann LJ, Gavilanes AW, Paolillo P, Fanos V, Caboni P, Barberini L, Noto A, Atzori L: Metabolomic analysis of bronchoalveolar lavage fluid in preterm infants complicated by respiratory distress syndrome: preliminary results. J Matern Fetal Neonatal Med. 2011;24:55–58. [18] Chu WL, Chu M, Wang YD, Hu Y, Zhao C, Su L, Xiong Y, Yang TS, Tao YH, Li HC: Effects of epithelial cell injury of the lower respiratory tract in the pathogenesis of allergic responses in a rat model. Chin Med J (Engl). 2013;126:72–77. [19] Ogino K, Zhang R, Takahashi H, Takemoto K, Kubo M, Murakami I, Wang DH, Fujikura Y: Allergic airway inflammation by nasal inoculation of particulate matter (PM2.5) in NC/Nga mice. PLoS One. 2014;9:e92710. doi: 10.1371/journal.pone.0092710. eCollection 2014. [20] Fricker M, Deane A, Hansbro PM: Animal models of chronic obstructive pulmonary disease. Expert Opin Drug Discov. 2014;9:629–645. [21] Martin JG, Tamaoka M: Rat models of asthma and chronic obstructive lung disease. Pulm Pharmacol Ther. 2006;19: 377–385. [22] Seferian A, Simonneau G: Therapies for pulmonary arterial hypertension: where are we today, where do we go tomorrow? Eur Respir Rev. 2013;22:217–226. [23] National Heart, Blood and Lung Institute, National Institutes of Health. Types of pulmonary hypertension. URL: http:// www.nhlbi.nih.gov/health/health-topics/topics/pah/types.html Accessed on 3rd June 2014 [24] Ho WE, Xu YJ, Cheng C, Peh HY, Tannenbaum SR, Wong WS, Ong CN: Metabolomics reveals inflammatory-linked pulmonary metabolic alterations in a murine model of house dust mite-induced allergic asthma. J Proteome Res. 2014 Jul 2. [Epub ahead of print] [25] Ami D, Natalello A, Doglia SM: Fourier transform infrared microspectroscopy of complex biological systems: from intact cells to whole organisms. Methods Mol Biol. 2012;895:85–100. [26] Neujahr DC, Uppal K, Force SD, Fernandez F, Lawrence C, Pickens A, Bag R, Lockard C, Kirk AD, Tran V, Lee K, Jones DP, Park Y: Bile acid aspiration associated with lung chemical profile linked to other biomarkers of injury after lung transplantation. Am J Transplant. 2014;14:841–888. [27] Wolak JE, Esther CR Jr, O’Connell TM: Metabolomic analysis of bronchoalveolar lavage fluid from cystic fibrosis patients. Biomarkers. 2009;14:55–60.

Experimental Lung Research

Metabolomic analysis of lung epithelial secretions in rats: an investigation of bronchoalveolar lavage fluid by GC-MS and FT-IR.

Rat bronchoalveolar lavage fluid (BALF) metabolome can be used to obtain valuable, precise, and accurate information about underlying lung conditions ...
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