Application of Mass Spectrometry-Based Metabolomics in Identification of Early Noninvasive Biomarkers of Alcohol-Induced Liver Disease Using Mouse Model

  • Soumen K. Manna
  • Matthew D. Thompson
  • Frank J. GonzalezEmail author
Conference paper
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 815)


A rapid, non-invasive urine test for early stage alcohol-induced liver disease (ALD) would permit risk stratification and treatment of high-risk individuals before ALD leads to irreversible liver damage and death. Urinary metabolomic studies were carried out to identify ALD-associated metabolic biomarkers using Ppara-null mouse model that is susceptible to ALD development on chronic alcohol consumption. Two successive studies were conducted to evaluate the applicability of mass spectrometry-based metabolomics in identification of ALD-specific signatures and to examine the robustness of these biomarkers against genetic background. Principal components analysis of ultraperformance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS)-generated urinary metabolic fingerprints showed that alcohol-treated wild-type and Ppara-null mice could be distinguished from control animals. It also showed that a combined endogenous biomarker panel helps to identify subjects with ALD as well as those at risk of developing ALD even without any information on alcohol intake or genetics. Quantitative analysis showed that increased excretion of indole-3-lactic acid and phenyllactic acid was a genetic background-independent signature exclusively associated with ALD pathogenesis in Ppara-null mice that showed liver pathologies similar to those observed in early stages of human ALD. These findings demonstrated that mass spectrometry-based metabolomic analysis could help in the identification of ALD-specific signatures, and that metabolites such as indole-3-lactic acid and phenyllactic acid, may serve as robust noninvasive biomarkers for early stages of ALD.


Alcohol-induced liver disease PPARα Ppara-null mouse Steatosis Metabolomics UPLC-ESI-QTOFMS Multivariate data analysis Biomarker Genetic background Indole-3-lactic acid Phenyllactic acid 



Alcohol-induced liver disease


Alanine aminotransferase


Analysis of variance


Aspartate aminotransferase


Electrospray ionization in positive mode


Electrospray ionization in negative mode


Multiple reaction monitoring


Oxidized nicotinamide adenine dinucleotide


Reduced nicotinamide adenine dinucleotide


Orthogonal projection to latent structures


Principal components analysis


Peroxisome proliferator-activated receptor alpha knockout mouse model


Peroxisome proliferator-activated receptor alpha


Ultraperformance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry



This work was supported by the National Cancer Institute Intramural Research Program, the National Institute of Environmental Health Sciences grant (U01ES016013).


  1. 1.
    Hoyert DL, Heron MP, Murphy SL, Kung HC (2006) Deaths: final data for 2003. Natl Vital Stat Rep 54(13):1–120PubMedGoogle Scholar
  2. 2.
    Mandayam S, Jamal MM, Morgan TR (2004) Epidemiology of alcoholic liver disease. Semin Liver Dis 24(3):217–232PubMedCrossRefGoogle Scholar
  3. 3.
    WHO (2011) Global status report on alcohol and health. World Health Organization, GenevaGoogle Scholar
  4. 4.
    Shibuya A, Yoshida A (1988) Genotypes of alcohol-metabolizing enzymes in Japanese with alcohol liver diseases: a strong association of the usual Caucasian-type aldehyde dehydrogenase gene (ALDH1(2)) with the disease. Am J Hum Genet 43(5):744–748PubMedCentralPubMedGoogle Scholar
  5. 5.
    Pirmohamed M, Kitteringham NR, Quest LJ et al (1995) Genetic polymorphism of cytochrome P4502E1 and risk of alcoholic liver disease in Caucasians. Pharmacogenetics 5(6):351–357PubMedCrossRefGoogle Scholar
  6. 6.
    Tanaka F, Shiratori Y, Yokosuka O, Imazeki F, Tsukada Y, Omata M (1996) High incidence of ADH2*1/ALDH2*1 genes among Japanese alcohol dependents and patients with alcoholic liver disease. Hepatology 23(2):234–239PubMedCrossRefGoogle Scholar
  7. 7.
    Zintzaras E, Stefanidis I, Santos M, Vidal F (2006) Do alcohol-metabolizing enzyme gene polymorphisms increase the risk of alcoholism and alcoholic liver disease? Hepatology 43(2):352–361PubMedCrossRefGoogle Scholar
  8. 8.
    Sherva R, Rice JP, Neuman RJ, Rochberg N, Saccone NL, Bierut LJ (2009) Associations and interactions between SNPs in the alcohol metabolizing genes and alcoholism phenotypes in European Americans. Alcohol Clin Exp Res 33(5):848–857PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Auguet T, Vidal F, Broch M et al (2010) Polymorphisms in the interleukin-10 gene promoter and the risk of alcoholism and alcoholic liver disease in Caucasian Spaniard men. Alcohol 44(3):211–216PubMedCrossRefGoogle Scholar
  10. 10.
    Linneberg A, Gonzalez-Quintela A, Vidal C et al (2010) Genetic determinants of both ethanol and acetaldehyde metabolism influence alcohol hypersensitivity and drinking behaviour among Scandinavians. Clin Exp Allergy 40(1):123–130PubMedCrossRefGoogle Scholar
  11. 11.
    O’Shea RS, Dasarathy S, McCullough AJ (2010) Alcoholic liver disease. Am J Gastroenterol 105(1):14–32; quiz 33PubMedCrossRefGoogle Scholar
  12. 12.
    Chen AC, Manz N, Tang Y et al (2010) Single-nucleotide polymorphisms in corticotropin releasing hormone receptor 1 gene (CRHR1) are associated with quantitative trait of event-related potential and alcohol dependence. Alcohol Clin Exp Res 34(6):988–996PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Grove J, Brown AS, Daly AK, Bassendine MF, James OF, Day CP (1998) The RsaI polymorphism of CYP2E1 and susceptibility to alcoholic liver disease in Caucasians: effect on age of presentation and dependence on alcohol dehydrogenase genotype. Pharmacogenetics 8(4):335–342PubMedCrossRefGoogle Scholar
  14. 14.
    Wong NA, Rae F, Bathgate A, Smith CA, Harrison DJ (2000) Polymorphisms of the gene for microsomal epoxide hydrolase and susceptibility to alcoholic liver disease and hepatocellular carcinoma in a Caucasian population. Toxicol Lett 115(1):17–22PubMedCrossRefGoogle Scholar
  15. 15.
    Teli MR, Day CP, Burt AD, Bennett MK, James OF (1995) Determinants of progression to cirrhosis or fibrosis in pure alcoholic fatty liver. Lancet 346(8981):987–990PubMedCrossRefGoogle Scholar
  16. 16.
    MacSween RN, Burt AD (1986) Histologic spectrum of alcoholic liver disease. Semin Liver Dis 6(3):221–232PubMedCrossRefGoogle Scholar
  17. 17.
    Schutte K, Bornschein J, Malfertheiner P (2009) Hepatocellular carcinoma—epidemiological trends and risk factors. Dig Dis 27(2):80–92PubMedCrossRefGoogle Scholar
  18. 18.
    Sherman M (2010) Hepatocellular carcinoma: New and emerging risks. Dig Liver Dis 42(Suppl 3):S215–S222PubMedCrossRefGoogle Scholar
  19. 19.
    Levitsky J, Mailliard ME (2004) Diagnosis and therapy of alcoholic liver disease. Semin Liver Dis 24(3):233–247PubMedCrossRefGoogle Scholar
  20. 20.
    Menon KV, Gores GJ, Shah VH (2001) Pathogenesis, diagnosis, and treatment of alcoholic liver disease. Mayo Clin Proc 76(10):1021–1029PubMedCrossRefGoogle Scholar
  21. 21.
    Crabb DW, Liangpunsakul S (2006) Alcohol and lipid metabolism. J Gastroenterol Hepatol 21(Suppl 3):S56–S60PubMedCrossRefGoogle Scholar
  22. 22.
    Lee SS, Pineau T, Drago J et al (1995) Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol Cell Biol 15(6):3012–3022PubMedCentralPubMedGoogle Scholar
  23. 23.
    Martin PG, Guillou H, Lasserre F et al (2007) Novel aspects of PPARalpha-mediated regulation of lipid and xenobiotic metabolism revealed through a nutrigenomic study. Hepatology 45(3):767–777PubMedCrossRefGoogle Scholar
  24. 24.
    Rakhshandehroo M, Sanderson LM, Matilainen M et al (2007) Comprehensive analysis of PPARalpha-dependent regulation of hepatic lipid metabolism by expression profiling. PPAR Res 2007:26839PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Sozio M, Crabb DW (2008) Alcohol and lipid metabolism. Am J Physiol Endocrinol Metab 295(1):E10–E16PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Nakajima T, Kamijo Y, Tanaka N et al (2004) Peroxisome proliferator-activated receptor alpha protects against alcohol-induced liver damage. Hepatology 40(4):972–980PubMedCrossRefGoogle Scholar
  27. 27.
    Mancinelli R, Ceccanti M (2009) Biomarkers in alcohol misuse: their role in the prevention and detection of thiamine deficiency. Alcohol Alcohol 44(2):177–182PubMedCrossRefGoogle Scholar
  28. 28.
    Sharpe PC (2001) Biochemical detection and monitoring of alcohol abuse and abstinence. Ann Clin Biochem 38(Pt 6):652–664PubMedCrossRefGoogle Scholar
  29. 29.
    Saadeh S (2007) Nonalcoholic Fatty liver disease and obesity. Nutr Clin Pract 22(1):1–10PubMedCrossRefGoogle Scholar
  30. 30.
    Calvaruso V, Craxi A (2009) Implication of normal liver enzymes in liver disease. J Viral Hepat 16(8):529–536PubMedCrossRefGoogle Scholar
  31. 31.
    Cadranel JF, Rufat P, Degos F (2000) Practices of liver biopsy in France: results of a prospective nationwide survey. For the Group of Epidemiology of the French Association for the Study of the Liver (AFEF). Hepatology 32(3):477–481PubMedCrossRefGoogle Scholar
  32. 32.
    Tyburski JB, Patterson AD, Krausz KW et al (2008) Radiation metabolomics. 1. Identification of minimally invasive urine biomarkers for gamma-radiation exposure in mice. Radiat Res 170(1):1–14PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Patterson AD, Lanz C, Gonzalez FJ, Idle JR (2009) The role of mass spectrometry-based metabolomics in medical countermeasures against radiation. Mass Spectrom Rev 29(3):503–521Google Scholar
  34. 34.
    Sreekumar A, Poisson LM, Rajendiran TM et al (2009) Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression. Nature 457(7231):910–914PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    MacIntyre DA, Jimenez B, Lewintre EJ et al (2010) Serum metabolome analysis by 1H-NMR reveals differences between chronic lymphocytic leukaemia molecular subgroups. Leukemia 24(4):788–797PubMedCrossRefGoogle Scholar
  36. 36.
    Loftus N, Barnes A, Ashton S et al (2011) Metabonomic investigation of liver profiles of nonpolar metabolites obtained from alcohol-dosed rats and mice using high mass accuracy MSn analysis. J Proteome Res 10(2):705–713PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Bradford BU, O’Connell TM, Han J et al (2008) Metabolomic profiling of a modified alcohol liquid diet model for liver injury in the mouse uncovers new markers of disease. Toxicol Appl Pharmacol 232(2):236–243PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Fernando H, Kondraganti S, Bhopale KK et al (2010) (1)H and (3)(1)P NMR lipidome of ethanol-induced fatty liver. Alcohol Clin Exp Res 34(11):1937–1947PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Pearson K (1901) On lines and planes of closest fit to systems of points in space. Philos Mag 2(6):559–572CrossRefGoogle Scholar
  40. 40.
    Suhre K, Schmitt-Kopplin P (2008) MassTRIX: mass translator into pathways. Nucleic Acids Res 36(Web Server Issue):W481–W484PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Jansson J, Willing B, Lucio M et al (2009) Metabolomics reveals metabolic biomarkers of Crohn’s disease. PLoS One 4(7):e6386PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Kind T, Fiehn O (2007) Seven Golden Rules for heuristic filtering of molecular formulas obtained by accurate mass spectrometry. BMC Bioinformatics 8:105PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Cui Q, Lewis IA, Hegeman AD et al (2008) Metabolite identification via the Madison Metabolomics Consortium Database. Nat Biotechnol 26(2):162–164PubMedCrossRefGoogle Scholar
  44. 44.
    Smith CA, O’Maille G, Want EJ et al (2005) METLIN: a metabolite mass spectral database. Ther Drug Monit 27(6):747–751PubMedCrossRefGoogle Scholar
  45. 45.
    Nguyen PV, Abel T, Kandel ER, Bourtchouladze R (2000) Strain-dependent differences in LTP and hippocampus-dependent memory in inbred mice. Learn Mem 7(3):170–179PubMedCentralPubMedCrossRefGoogle Scholar
  46. 46.
    Syn WK, Yang L, Chiang DJ et al (2009) Genetic differences in oxidative stress and inflammatory responses to diet-induced obesity do not alter liver fibrosis in mice. Liver Int 29(8):1262–1272PubMedCentralPubMedCrossRefGoogle Scholar
  47. 47.
    Liu J, Corton C, Dix DJ, Liu Y, Waalkes MP, Klaassen CD (2001) Genetic background but not metallothionein phenotype dictates sensitivity to cadmium-induced testicular injury in mice. Toxicol Appl Pharmacol 176(1):1–9PubMedCrossRefGoogle Scholar
  48. 48.
    Manna SK, Patterson AD, Yang Q et al (2010) Identification of noninvasive biomarkers for alcohol-induced liver disease using urinary metabolomics and the Ppara-null mouse. J Proteome Res 9(8):4176–4188PubMedCentralPubMedCrossRefGoogle Scholar
  49. 49.
    Manna SK, Patterson AD, Yang Q et al (2011) UPLC–MS-based urine metabolomics reveals indole-3-lactic acid and phenyllactic acid as conserved biomarkers for alcohol-induced liver disease in the Ppara-null mouse model. J Proteome Res 10(9):4120–4133PubMedCentralPubMedCrossRefGoogle Scholar
  50. 50.
    Helander A, Bottcher M, Fehr C, Dahmen N, Beck O (2009) Detection times for urinary ethyl glucuronide and ethyl sulfate in heavy drinkers during alcohol detoxification. Alcohol Alcohol 44(1):55–61PubMedCrossRefGoogle Scholar
  51. 51.
    Hoiseth G, Bernard JP, Stephanson N et al (2008) Comparison between the urinary alcohol markers EtG, EtS, and GTOL/5-HIAA in a controlled drinking experiment. Alcohol Alcohol 43(2):187–191PubMedCrossRefGoogle Scholar
  52. 52.
    Shin M, Kim I, Inoue Y, Kimura S, Gonzalez FJ (2006) Regulation of mouse hepatic alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase, a key enzyme in the tryptophan-nicotinamide adenine dinucleotide pathway, by hepatocyte nuclear factor 4alpha and peroxisome proliferator-activated receptor alpha. Mol Pharmacol 70(4):1281–1290PubMedCrossRefGoogle Scholar
  53. 53.
    Lumeng L, Crabb DW (2001) Alcoholic liver disease. Curr Opin Gastroenterol 17(3):211–220PubMedCrossRefGoogle Scholar
  54. 54.
    Kalant H, Khanna JM, Loth J (1970) Effect of chronic intake of ethanol on pyridine nucleotide levels in rat liver and kidney. Can J Physiol Pharmacol 48(8):542–549PubMedCrossRefGoogle Scholar
  55. 55.
    Recasens M, Benezra R, Basset P, Mandel P (1980) Cysteine sulfinate aminotransferase and aspartate aminotransferase isoenzymes of rat brain. Purification, characterization, and further evidence for identity. Biochemistry 19(20):4583–4589PubMedCrossRefGoogle Scholar
  56. 56.
    Yagi T, Kagamiyama H, Motosugi K, Nozaki M, Soda K (1979) Crystallization and properties of aspartate aminotransferase from Escherichia coli B. FEBS Lett 100(1):81–84PubMedCrossRefGoogle Scholar
  57. 57.
    Jean M, DeMoss RD (1968) Indolelactate dehydrogenase from Clostridium sporogenes. Can J Microbiol 14(4):429–435PubMedCrossRefGoogle Scholar
  58. 58.
    Leelayoova S, Marbury D, Rainey PM, Mackenzie NE, Hall JE (1992) In vitro tryptophan catabolism by Leishmania donovani donovani promastigotes. J Protozool 39(2):350–358PubMedCrossRefGoogle Scholar
  59. 59.
    Owen TG, Hochachka PW (1974) Purification and properties of dolphin muscle aspartate and alanine transaminases and their possible roles in the energy metabolism of diving mammals. Biochem J 143(3):541–553PubMedCentralPubMedGoogle Scholar
  60. 60.
    Bode R, Lippoldt A, Birnbaum D (1986) Purification and properties of D-aromatic lactate dehydrogenase, an enzyme involved in the catabolism of the aromatic amino acids of Candida maltosa. Biochem Physiol Pflanz 181:189–198CrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Soumen K. Manna
    • 1
  • Matthew D. Thompson
    • 1
  • Frank J. Gonzalez
    • 1
    Email author
  1. 1.Laboratory of Metabolism, Center for Cancer ResearchNational Cancer InstituteBethesdaUSA

Personalised recommendations