Chromatographia

, Volume 75, Issue 21–22, pp 1301–1310 | Cite as

GC/TOFMS Analysis of Endogenous Metabolites in Mouse Fibroblast Cells and Its Application in TiO2 Nanoparticle-Induced Cytotoxicity Study

  • Yumin Liu
  • Yu Cheng
  • Tianlu Chen
  • Yinan Zhang
  • Xiaoyan Wang
  • Aihua Zhao
  • Wei Jia
  • Yang Bo
  • Chengyu Jin
Original
First Online:
Received:
Revised:
Accepted:

Abstract

In this paper, we present an optimized procedure for metabolomic analysis of endogenous metabolites in mouse fibroblast (L929) cell line using gas chromatography/time-of-flight mass spectrometry with multivariate statistics. The optimization of metabolite extraction was performed using three solvents: methanol, water, and chloroform, and then followed by methoxymation and silylation. This method was subsequently validated using 29 reference standards and cell line samples. The intra- and inter-day relative standard deviations (RSDs) of the standard compounds were lower than 15.0 and 25.0 %, respectively. As for most of the tested metabolites in cell line samples, RSDs were below 20.0 % for reproducibility and stability, respectively. We applied this approach in metabolomic study of L929 cells obtained from TiO2 nanoparticle-induced cytotoxicity model samples (n = 5) and control samples (n = 5). Metabolite markers associated with TiO2 nanoparticle-induced cytotoxicity were identified and validated by statistical methods and reference standards. Our work highlights the potential of this method for cell metabolomic study.

Keywords

Gas chromatography/time-of-flight mass spectrometry TiO2 nanoparticle Metabolomics Mouse fibroblast cell 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

This work was financially supported by Shanghai Natural Science Foundation of the Science and Technology Commission of Shanghai Municipal Government (No. 09ZR1415100), two Fundamental Key Project (No. YG2010MS92; YG2011MS66) of Shanghai Jiaotong University.

Supplementary material

10337_2012_2315_MOESM1_ESM.doc (73 kb)
Supplementary material 1 (DOC 73 kb)

References

  1. 1.
    Fiehn O (2002) Metabolomics–the link between genotypes and phenotypes. Plant Mol Biol 48:155–171CrossRefGoogle Scholar
  2. 2.
    Vulimiri SV, Berger A, Sonawane B (2011) The potential of metabolomic approaches for investigating mode(s) of action of xenobiotics: case study with carbon tetrachloride. Mutat Res 722(2):147–153CrossRefGoogle Scholar
  3. 3.
    Lindon JC, Holmes E, Bollard ME, Stanley EG, Nicholson JK (2004) Metabonomics technologies and their applications in physiological monitoring, drug safety assessment and disease diagnosis. Biomarkers 9(1):1–31CrossRefGoogle Scholar
  4. 4.
    Brindle JT, Antti H, Holmes E, Tranter G, Nicholson JK, Bethell HWL, Clarke S, Schofield PM, McKilligin E, Mosedale DE, Grainger DJ (2002) Rapid and noninvasive diagnosis of the presence and severity of coronary heart disease using H-1 NMR-based metabonomics. Nat Med 8:1439–1445CrossRefGoogle Scholar
  5. 5.
    Jiye A, Trygg J, Gullberg J, Johansson AI, Jonsson P, Antti H, Marklund SL, Moritz T (2005) Extraction and GC/MS analysis of the human blood plasma metabolome. Anal Chem 77(24):8086–8094CrossRefGoogle Scholar
  6. 6.
    Bruce Stephen J, Tavazzi Isabelle, Parisod Veronique, Rezzi Serge, Kochhar Sunil, Guy Philippe A (2009) Investigation of human blood plasma sample preparation for performing metabolomics using ultrahigh performance liquid chromatography/mass spectrometry. Anal Chem 81(9):3285–3296CrossRefGoogle Scholar
  7. 7.
    Ramautar R, Mayboroda OA, Somsen GW, de Jong GJ (2011) CE-MS for metabolomics: developments and applications in the period 2008–2010. Electrophoresis 32:52–65CrossRefGoogle Scholar
  8. 8.
    Beckonert O, Keun HC, Ebbels TM et al (2007) Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts. Nat Protoc 2(11):2692–2703CrossRefGoogle Scholar
  9. 9.
    Villas-Bôas SG, Mas S, Akesson M, Smedsgaard J, Nielsen J (2005) Mass spectrometry in metabolome analysis. Mass Spectrom Rev 24:613–646CrossRefGoogle Scholar
  10. 10.
    Qiu Y, Su M, Liu Y, Chen M, Gu J, Zhang J, Jia W (2007) Application of ethyl chloroformate derivatization for gas chromatography-mass spectrometry based metabonomic profiling. Anal Chim Acta 583:277–283CrossRefGoogle Scholar
  11. 11.
    Liu YM, Chen TL, Qiu YP, Cheng Y, Cao Y, Zhao AH, Jia W (2011) An ultrasonication-assisted extraction and derivatization protocol for GC/TOFMS-based metabolite profiling. Anal Bioanal Chem 400(5):1405–1417CrossRefGoogle Scholar
  12. 12.
    Pan L, Qiu YP, Chen TL, Lin JC, Chi Y, Su MM, Zhao AH, Jia W (2010) An optimized procedure for metabonomic analysis of rat liver tissue using gas chromatography/time-of-flight mass spectrometry. J Pharm Biomed Anal 52(4):589–596CrossRefGoogle Scholar
  13. 13.
    Jin CY, Zhu BS, Wang XF, Lu QH (2008) Cytotoxicity of titanium dioxide nanoparticles in mouse fibroblast cells. Chem Res Toxicol 21(9):1871–1877CrossRefGoogle Scholar
  14. 14.
    Arora S, Jain J, Rajwade JM, Paknikar KM (2009) Interactions of silver nanoparticles with primary mouse fibroblasts and liver cells. Toxicol Appl Pharmacol 236(3):310–318CrossRefGoogle Scholar
  15. 15.
    Zhang Y, Jiye A, Wang G, Huang Q, Yan B, Zha W, Gu S, Liu L, Ren H, Ren M, Sheng L (2009) Organic solvent extraction and metabonomic profiling of the metabolites in erythrocytes. J Chromatogr B Analyt Technol Biomed Life Sci 877(18–19):1751–1757Google Scholar
  16. 16.
    Sellick CA, Knight D, Croxford AS, Maqsood AR, Stephens GM, Goodacre R, Dickson AJ (2010) Evaluation of extraction processes for intracellular metabolite profiling of mammalian cells: matching extraction approaches to cell type and metabolite targets. Metabolomics 6:427–438CrossRefGoogle Scholar
  17. 17.
    Anselmann R (2001) Nanoparticles and nanolayers in commercial applications. J Nanoparticle Res 3:329–336CrossRefGoogle Scholar
  18. 18.
    Doumanidis H (2002) The nanomanufacturing programme at the National Science Foundation. Nanotechnology 13:248–252CrossRefGoogle Scholar
  19. 19.
    Tsuji JS, Maynard AD, Howard PC, James JT, Lam C, Warheit DB, Santamariak AB (2006) Research strategies for safety evaluation of nanomaterials, part IV: risk assessment of nanoparticles. Toxicol Sci 89:42–50CrossRefGoogle Scholar
  20. 20.
    Warheit DB, Webb TR, Reed KL, Frerichs S, Sayes CM (2007) Pulmonary toxicity study in rats with three forms of ultrafine-TiO2 particles: differential responses related to surface properties. Toxicology 230:90–104CrossRefGoogle Scholar
  21. 21.
    Soto K, Garza KM, Murr LE (2007) Cytotoxic effects of aggregated nanomaterials. Acta Biomater 3:351–358CrossRefGoogle Scholar
  22. 22.
    Bennett BD, Yuan J, Kimball EH, Rabinowitz JD (2008) Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach. Nat Protoc 3(8):1299–1311CrossRefGoogle Scholar
  23. 23.
    Jonsson P, Gullberg J, Nordstrom A et al (2004) Design of experiments: an efficient strategy to identify factors influencing extraction and derivatization of Arabidopsis thaliana samples in metabolomic studies with gas chromatography/mass spectrometry. Anal Chem 76:1738–1745CrossRefGoogle Scholar
  24. 24.
    Jonsson P, Johansson A, Gullberg J et al (2005) High-throughput data analysis for detecting and identifying differences between samples in GC/MS-based metabolomic analyses. Anal Chem 77:5635–5642CrossRefGoogle Scholar
  25. 25.
    Wold S, Sjostrom M, Eriksson L (2001) PLS-regression: a basic tool of chemometrics. Chemom Intell Lab Syst 58:109–130CrossRefGoogle Scholar
  26. 26.
    Stenlund H, Gorzsas A, Persson P, Sundberg B, Trygg J (2008) Orthogonal projections to latent structures discriminant analysis modeling on in situ FT-IR spectral imaging of liver tissue for identifying sources of variability. Anal Chem 80:6898–6906CrossRefGoogle Scholar
  27. 27.
    Gao XF, Pujos-Guillot E, Sébédio JL (2010) Development of a quantitative metabolomic approach to study clinical human fecal water metabolome based on trimethylsilylation derivatization and GC/MS analysis. Anal Chem 82:6447–6456CrossRefGoogle Scholar
  28. 28.
    Tang M, Zhang T, Xue YY, Wang S, Huang MM, Yang Y, Lu MY, Lei H, Kong L, Wang YQ, Pu YP (2011) Metabonomic studies of biochemical changes in the serum of rats by intratracheally instilled TiO2 nanoparticles. J Nanosci Nanotechnol 11:3065–3074CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Yumin Liu
    • 1
    • 2
  • Yu Cheng
    • 4
  • Tianlu Chen
    • 3
  • Yinan Zhang
    • 1
  • Xiaoyan Wang
    • 3
  • Aihua Zhao
    • 1
  • Wei Jia
    • 5
  • Yang Bo
    • 2
  • Chengyu Jin
    • 2
  1. 1.School of PharmacyShanghai Jiao Tong UniversityShanghaiChina
  2. 2.Center for Instrumental AnalysisShanghai Jiao Tong UniversityShanghaiChina
  3. 3.Shanghai Center for Systems BiomedicineShanghai Jiao Tong UniversityShanghaiChina
  4. 4.Department of ChemistryEast China Normal UniversityShanghaiChina
  5. 5.Department of NutritionUniversity of North Carolina at Greensboro, North Carolina Research CampusKannapolisUSA

Personalised recommendations